Method for isolating cross-reactive aptamer and use thereof

ABSTRACT

The subject invention provides a SELEX strategy for isolating cross-reactive aptamers that recognize a core structure of a small-molecule family and bind to several structurally-similar molecules in the family. The subject invention also provides methods, assays, and products for detecting small-molecule targets of the family in a sample in both clinical and field settings. Such method is based on an aptamer sensor that reports the presence of small-molecule targets via a sensitive colorimetric signal for naked-eye detection. The subject invention further provides exonuclease-based methods for generating structure-switching aptamers from fully folded aptamers and developing electrochemical aptamer-based (E-AB) sensors for rapid and sensitive detection of synthetic cathinones.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a divisional application of co-pending U.S. Serialapplication Ser. No. 16/791,101, filed Feb. 14, 2020; which is acontinuation application of U.S. Serial application Ser. No. 16/174,764,filed Oct. 30, 2018, now U.S. Pat. No. 10,655,132; all of which areincorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under 2016-DN-BX-0167awarded by the National Institute of Justice. The government has certainrights in the invention.

SEQUENCE LISTING

The Sequence Listing for this application is labeled“SeqList-28Jun19-ST25.txt,” which was created on Jun. 28, 2019, and is 5KB. The Sequence Listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Small molecules are important targets with the potential of clinical orcommercial applications such as medical diagnostics, environmentalmonitoring, and forensic science. Thus, efforts to develop methods forportable, low-cost, point-of-care and quantitative detection of a broadrange of small molecules are gaining momentum.

Synthetic cathinones (also known as bath salts) are designer drugssharing a similar core structure with amphetamines and 3,4-methylenedioxy-methamphetamine (MDMA). They are highly addictivecentral nervous system stimulants, and are associated with many negativehealth consequences, including even death. Although these drugs haveemerged only recently, abuse of bath salts has become a threat to publichealth and safety due to their severe toxicity, increasingly broadavailability, and difficulty of regulation. More importantly, there iscurrently no reliable presumptive test for any synthetic cathinone.Chemical spot tests used to detect conventional drugs such as cocaine,methamphetamine, and opioids show no cross-reactivity to syntheticcathinones.

Screening for small molecules such as synthetic cathinones requirescross-reactive assays that can broadly detect small molecules based ontheir shared molecular framework. Such assays are more efficient andcost-effective than the tandem use of multiple highly specific assaysthat detect a single analyte.

Antibody-based immunoassays have dominated the field of on-sitesmall-molecule detection, and while numerous assays have been developedfor a variety of individual targets, the development of cross-reactiveimmunoassays has proven difficult. This is in part because the processof antibody generation, which is entirely in vivo, provides no controlover the cross-reactivity of the generated antibody.

Nucleic acid-based bioaffinity elements known as aptamers hold muchpromise in overcoming many of the shortcomings associated withimmunoassays. Aptamers are isolated through a process known assystematic evolution of ligands by exponential enrichment (SELEX) tobind targets of interest with high affinity and specificity. Aptamerscan be isolated for essentially any target, including metal ions, smallmolecules, proteins, or whole cells.

Unlike antibodies, aptamers can be isolated relatively quickly andchemically synthesized in an inexpensive manner with no batch-to-batchvariation. Aptamers are chemically stable and have shelf-lives of a fewyears at room temperature. Moreover, aptamers can be engineered to havetunable target-binding affinities or various functionalities. Theseadvantages make aptamers ideal for use in biosensors.

Because SELEX is an in vitro process, it should be possible to isolate across-reactive aptamer through precise control of the selection strategyand conditions. Ideally, such aptamer should bind to the core structureof a given class of targets while being insensitive to peripheralsubstituents, thereby capable of recognizing the whole target family.However, little work has been done to demonstrate the capability ofSELEX to achieve such goal.

Therefore, there is a need for developing a novel SELEX strategy toisolate cross-reactive aptamers for structurally-similar compounds.There are also needs for methods and materials for rapid, sensitive,on-site, and naked-eye detection of small molecules such as syntheticcathinones.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides a novel SELEX strategy for isolatingcross-reactive aptamers that recognize a core structure of asmall-molecule family and bind each structurally-similar molecule insaid family.

In one embodiment, the method employs a parallel-and-serial SELEXstrategy, comprising at least one step of a parallel selection and atleast one step of a serial selection. In another embodiment, the methodfor isolating an aptamer for a family of structurally-similar smallmolecules comprises mixing the small molecules in said family with anucleic acid library, binding the small molecules to one or moreaptamers in the nucleic acid library, separating the aptamer bound tothe small molecules in said family from at least a portion of theunbound nucleic acid molecules, isolating the aptamer and optionally,amplifying the isolated aptamer.

In one embodiment, the aptamer isolated by the method according to thesubject invention is a cross-reactive aptamer that recognizes and bindsto the core structure of synthetic cathinones. The synthetic cathinonesinclude, but are not limited to MDPV, ethylone, naphyrone, penthylone,methylone, buthylone, MPHP, 4-MMC, methedrone, pyrovalenrone, MDPBP,α-PVP, MEPBP, 4-FMC, and methcathinone. The aptamer does not cross-reactwith common cutting agents found in seized samples such aspseudoephedrine, promazine, procaine, ephedrine, acetaminophen,methamphetamine, lidocaine, amphetamine, cocaine, sucrose, and caffeine.

In one embodiment, the cross-reactive aptamer, according to thesubjection, is a DNA aptamer comprising at least 46 nucleotides. Thetarget-binding domain of the cross-reactive aptamer comprises anucleotide sequence selected from SEQ ID Nos: 7-17 and sequences sharingat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% identity with SEQ ID Nos: 7-17. In a specific embodiment, thecross-reactive aptamer comprises a nucleotide sequence of SCA2.1 (SEQ IDNO: 6). In a preferred embodiment, the cross-reactive aptamer is SCA2.1(SEQ ID NO: 6) and sequences sharing at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SCA2.1 (SEQID NO: 6).

The subject invention provides methods, assays, and products for rapid,naked-eye detection of small molecules in a sample, in particular, inboth clinical and field settings. The subject invention is based on anaptamer sensor that reports the presence of small-molecule targets suchas synthetic cathinones via a sensitive colorimetric signal fornaked-eye detection. Specifically, exemplified herein is a method fordetecting synthetic cathinones in bodily fluids, drinks, and/or seizedsubstances.

In one embodiment, the subject invention provides a method forgenerating cross-reactive aptamers with structure-switchingfunctionality and a means of rapid and sensitive detection of asmall-molecule target family. The generation of a structure-switchingcross-reactive aptamer entails digesting the cross-reactive aptamer withan exonuclease mixture, such as exonuclease III (Exo III) andexonuclease I (Exo I). The resulting digestion product hasstructure-switching functionality with similar or equal affinity as itsparent cross-reactive aptamer, and such structure-switchingcross-reactive aptamer can be directly employed in folding-based aptamersensors.

In some embodiments, the cross-reactive aptamers withstructure-switching functionality are fragments of the cross-reactiveaptamers. In specific embodiments, the structure-switchingcross-reactive aptamer comprises a nucleotide sequence of SCA-SW-40 (SEQID NO: 18) or SCA-SW-34 (SEQ ID NO: 19). Preferably, thestructure-switching cross-reactive aptamer is SCA-SW-40 (SEQ ID NO: 18),SCA-SW-34 (SEQ ID NO: 19) or sequences sharing at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SCA-SW-40 (SEQ IDNO: 18) or SCA-SW-34 (SEQ ID NO: 19).

In one embodiment, the subject invention provides a method for detectinga small molecule target or a small-molecule target family in a sampleusing the structure-switching cross-reactive aptamer.

In one embodiment, the subject invention provides methods for rapid andsensitive detection of a small-molecule target or a small-moleculetarget family in a sample by incorporating the structure-switchingcross-reactive aptamers into an electrochemical aptamer-based (E-AB)sensor, which has demonstrated target-induced conformational changeswithin the aptamers and has achieved excellent sensor performance. Themethod comprises contacting the sample with the E-AB sensor, anddetecting the small-molecule target/target family in the sample, whereinthe detection comprises measuring a signal generated from a signalreporter.

In one embodiment, the subject invention provides a kit suitable forscreening aptamers that bind to a family of structurally similarmolecules of interest. The kit comprises a suitable container, anoligonucleotide library, and instructions for use in performing a screenfor aptamers that bind to the family of structurally similar moleculesof interest. The kit may optionally further comprise one or morereagents, one or more suitable primers and enzymes, e.g., nucleases, andone or more buffers.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings(s) will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1B show the isolation of a cross-reactive aptamer binding tosynthetic cathinones using a parallel-and-serial SELEX strategy. (1A)The core structure of synthetic cathinones and the three targets chosenfor parallel-and-serial SELEX. The substituent moieties on the beta-ketophenethylamine core structure are shaded in red. (1B) The schematicdiagram of parallel-and-serial SELEX.

FIGS. 2A-2B show the determination of the target-binding affinity,cross-reactivity, and specificity of the round P5 ethylone pool via agel elution assay. (2A) Polyacrylamide gel electrophoresis (PAGE)results depict the target elution profile with lanes representingsamples of the pool eluted with 0, 50, 100, 250, 500, or 1000 μMethylone (from left to right). The percent of target-eluted pool wasplotted against the concentration of ethylone employed for elution todetermine the binding affinity of the enriched pool. (2B) PAGE resultsof enriched pool eluted with 500 μM synthetic cathinones (α-PVP,ethylone, and butylone) or interferents (cocaine, procaine, andlidocaine) was used to measure target-cross-reactivity and specificityof the enriched pool.

FIGS. 3A-3B show the determination of the target-binding affinity,cross-reactivity, and specificity of the round P5 butylone pool via agel elution assay. (3A) Polyacrylamide gel electrophoresis (PAGE)results depict the target elution profile with lanes representingsamples of the pool eluted with 0, 50, 100, 250, 500, or 1000 μMbutylone (from left to right). The percent of target-eluted pool wasplotted against the concentration of butylone employed for elution todetermine the binding affinity of the enriched pool. (3B) PAGE resultsof enriched pool eluted with 500 μM synthetic cathinones (α-PVP,ethylone, and butylone) or interferents (cocaine, procaine, andlidocaine) was used to measure target-cross-reactivity and specificityof the enriched pool.

FIGS. 4A-4B show the determination of the target-binding affinity,cross-reactivity, and specificity of the round P5 α-PVP pool via a gelelution assay. (4A) Polyacrylamide gel electrophoresis (PAGE) resultsdepict the target elution profile with lanes representing samples of thepool eluted with 0, 50, 100, 250, 500, or 1000 μM α-PVP (from left toright). The percent of target-eluted pool was plotted against theconcentration of α-PVP employed for elution to determine the bindingaffinity of the enriched pool. (4B) PAGE results of enriched pool elutedwith 500 μM synthetic cathinones (α-PVP, ethylone, and butylone) orinterferents (cocaine, procaine, and lidocaine) was used to measuretarget-cross-reactivity and specificity of the enriched pool.

FIGS. 5A-5B show the determination of the target-binding affinity,cross-reactivity, and specificity of the round P9 α-PVP pool via a gelelution assay. (5A) Polyacrylamide gel electrophoresis (PAGE) resultsdepict the target elution profile with lanes representing samples of thepool eluted with 0, 50, 100, 250, 500, or 1000 μM α-PVP (from left toright). The percent of target-eluted pool was plotted against theconcentration of α-PVP employed for elution to determine the bindingaffinity of the enriched pool. (5B) PAGE results of enriched pool elutedwith 500 μM synthetic cathinones (α-PVP, ethylone, and butylone) orinterferents (cocaine, procaine, and lidocaine) was used to measuretarget-cross-reactivity and specificity of the enriched pool.

FIGS. 6A-6B show the determination of the target-binding affinity,cross-reactivity and specificity of the round S3 pool via a gel elutionassay. (6A) PAGE results depict the target elution profile with lanesrepresenting samples of the pool eluted with 0, 10, 50, 100, 250, 500,or 1000 μM (from left to right) of α-PVP, ethylone, or butylone. Thepercent of target-eluted pool was plotted against the concentration oftarget used for elution to determine the binding affinity of theenriched pool. (6B) PAGE results of enriched pool eluted with 500 μMsynthetic cathinones (α-PVP, ethylone, and butylone) or interferents(cocaine, procaine, and lidocaine) was used to measuretarget-cross-reactivity and specificity of the enriched pool.

FIGS. 7A-7B show the determination of the target-binding affinity,cross-reactivity, and specificity of the final enriched pool via a gelelution assay. (7A) PAGE results depict the target elution profile withlanes representing samples of the pool eluted with 0, 10, 50, 100, 250,500, or 1000 μM (from left to right) α-PVP, ethylone, or butylone. Thepercent of target-eluted pool was plotted against the concentration oftarget to determine the binding affinity of the enriched pool. (7B)Percent elution values were reported for 16 synthetic cathinones and 11interferents at a concentration of 50 μM.

FIGS. 8A-8B show the chemical structures of the (8A) syntheticcathinones and (8B) interferent compounds studied in this work.

FIG. 9 shows the sequences of randomized region of oligonucleotides fromthe final enriched pool after trimming and alignment by BioEditsoftware. Sequences (SEQ ID NOs: 7-17) are oligonucleotides in therandomized region. The prevalence of each oligonucleotide is ranked fromhighest (top) to lowest (bottom) using the frequency of appearance(count) as a benchmark. SEQ ID NO: 17 is the oligonucleotide sequence ofthe randomized region of the SCA2.1 aptamer. SEQ ID NO: 16 is theoligonucleotide sequence of the randomized region of the SCA1.1 aptamer.SEQ ID NO: 15 is the oligonucleotide sequence of the randomized regionof the SCA1.2 aptamer.

FIG. 10 shows the secondary structure of SCA2.1 (SEQ ID NO: 6) aspredicted by Mfold under the selection ion concentrations (20 mM NaCland 0.5 mM MgCl₂) at 23° C. The estimated free energy of formation isshown below.

FIGS. 11A-11C show the characterization of the target-binding affinityof SCA2.1 using isothermal titration calorimetry. Top panels present rawdata showing the heat generated from each titration of (11A) butylone,(11B) ethylone and (11C) α-PVP to SCA2.1, while bottom panels show theintegrated heat of each titration after correcting for dilution heat ofthe titrant.

FIGS. 12A-12B show the colorimetric detection of synthetic cathinonesusing a Cy7-displacement assay. (12A) Schematic of the Cy7-displacementassay, wherein binding of a synthetic cathinone molecule displaces Cy7monomer from the binding domain of SCA2.1 (SEQ ID NO: 6), thus inducingformation of Cy7 dimer that produces a change in the absorbance of thedye. (12B) Calibration curves based on the absorbance ratio at 670/775nm in the presence of different concentrations of α-PVP, ethylone orbutylone (0, 0.1, 0.3, 0.5, 1.1, 2.3, 4.9, 10.3, 21.6, 45.4, 95.2, 200μM). The inset represents the linear range at 0 to 10 μM target. Errorbars show standard deviation from three measurements at eachconcentration. [SCA 2.1]=3 μM, [Cy7]=2 μM.

FIGS. 13A-13B show the determination of the binding affinity of Cy7 toSCA2.1 via a colorimetric assay. (13A) Absorbance spectra of 2 μM Cy7 inthe presence of varying concentrations of SCA2.1 (0.2, 0.4, 0.8, 1.6,3.1, 6.3, 12.5, or 25 μM), with the black-to-red color gradientrepresenting increasing concentrations of SCA2.1. (13B) The plot of theabsorbance of Cy7 monomer at its peak wavelength (775 nm) versusconcentration of SCA2.1 was fitted with the Langmuir equation todetermine the binding affinity of Cy7 to SCA2.1.

FIGS. 14A-14C show Cy7-displacement colorimetric assay for the detectionof synthetic cathinones using SCA2.1. Absorbance spectra of Cy7 (2 μM)in the presence of varying concentrations (0, 0.1, 0.3, 0.5, 1.1, 2.3,4.9, 10.3, 21.6, 45.4, 95.2, 200 μM) of (14A) butylone, (14B) ethylone,(14C) α-PVP, with the black-to-red color gradient representingincreasing concentrations of synthetic cathinone target. [SCA2.1]=3 μM.

FIGS. 15A-15B show the detection of ethylone in 50% urine using theSCA2.1-based Cy7-displacement colorimetric assay. (15A) Absorbancespectra of Cy7 (2 μM) in the presence of varying concentrations ofethylone (0, 0.03, 0.06, 0.12, 0.25, 0.53, 1.11, 2.33, 4.90, 10.28,21.60, 45.35, 95.24, 200 μM), with the black-to-red color gradientrepresenting increasing concentrations of synthetic cathinone target.(15B) Assay calibration curve generated using 0-200 μM ethylone with theinset representing linear range from 0 to 1.2 μM target. [SCA2.1]=3 μM.

FIG. 16A-16B show the detection of ethylone in 50% saliva using theSCA2.1-based Cy7-displacement colorimetric assay. (16A) Absorbancespectra of Cy7 (2 μM) in the presence of varying concentrations ofethylone (0, 0.03, 0.06, 0.12, 0.25, 0.53, 1.11, 2.33, 4.90, 10.28,21.60, 45.35, 95.24, 200 μM), with the black-to-red color gradientrepresenting increasing concentrations of synthetic cathinone target.(16B) Assay calibration curve generated using 0-200 μM ethylone with theinset representing linear range from 0 to 1.2 μM target. [SCA2.1]=3 μM.

FIGS. 17A-17B show the colorimetric detection of synthetic cathinonesusing a Cy7-displacement assay. (17A) Signal gain measured via aninstrument from the Cy7-displacement assay with 12 synthetic cathinones(gray, the three selection targets are shaded) and 11 commoninterferents (white) at a concentration of 50 μM with 3 μM SCA2.1 and 2μM Cy7. Error bars show standard deviations from three measurements ofeach compound. (17B) Naked-eye detection of synthetic cathinones using amixture of 5 μM of SCA2.1 and 3.5 μM Cy7. The solution appears as afaint blue color in the absence of target and 50 μM interferents (17B,a-i). However, the color of the solutions changes to bright blue withinseconds upon addition of 50 μM synthetic cathinones (17B, m-x).

FIG. 18 shows the calibration curve of naked-eye detection of ethylonein the concentration range of 0.4 μM to 200 μM. The blue color changecan be clearly observed with the ethylone concentration above 6.3 μM.

FIGS. 19A-19C show the exonuclease-mediated truncation of asynthetic-cathinone-binding aptamer to generate a structure-switchingaptamer. (19A) SCA2.1 (SEQ ID NO: 6) possesses a fully folded stem.Exonuclease-mediated truncation of SCA2.1 generates a (19B)structure-switching aptamer (SCA-SW-40, SEQ ID NO: 18) which exists inan unfolded single-stranded state in the absence of target, but (19C)folds into a double-stranded structure in the presence of syntheticcathinones.

FIGS. 20A-20C show the electrochemical aptamer-based sensing ofsynthetic cathinone drugs. The structure-switching aptamer SCA-SW-40 ismodified with a 6-carbon linker and a thiol group at its 5′-end, and amethylene blue redox tag at its 3′-end, respectively. (20A) In theabsence of target the immobilized aptamer remains in a single-strandedflexible state, orientating the redox tag away from the gold surfacewhich results in a small background current. (20B) In the presence of asynthetic cathinone drug, the aptamer binds to the target, transitioningto a double-stranded, folded structure which brings the redox tag closeto the electrode surface, (20C) resulting in a large current increase.

FIGS. 21A-21C show the effect of SCA-SW-40 surface coverage on E-ABsensor performance. (21A) The surface coverage of immobilized SCA-SW-40for each electrode was measured using chronocoulometry. (21B) MDPVcalibration curve was generated for each electrode (with differingSCA-SW-40 surface coverages) with a measurement frequency of 150 Hz.(21C) The stability of SCA-SW-40 monolayer on each electrode wasmeasured before and after 50 scans. Error bars shown are standard errorfor the mean of measurements using three separate electrodes.Experiments were performed in selection buffer (10 mM Tris-HCl (pH 7.4),20 mM NaCl, 0.5 mM MgCl₂).

FIGS. 22A-22B show the optimization of MgCl₂ and NaCl in E-AB sensorbuffer. (22A) Detection of 30 μM MDPV was performed under various MgCl₂concentrations in the absence of NaCl. (22B) Detection of 30 μM MDPV wasthen performed using the optimized MgCl₂ concentration with variousconcentrations of NaCl. (Surface coverage=1.5×10¹² molecules/cm²).

FIGS. 23A-23B show the optimization of SCA-SW-40 surface coverage forenhanced E-AB performance. Gold electrodes were modified with varyingconcentrations of SCA-SW-40 (10, 25, or 50 nM). (23A) The surfacecoverage of immobilized SCA-SW-40 was measured using chronocoulometry.(23B) A MDPV calibration curve was generated for each electrode with ameasurement frequency of 150 Hz. Error bars shown standard errors forthe mean of measurements using three separate electrodes. Experimentswere performed using the optimized E-AB sensor buffer (10 mM Tris-HCl(pH 7.4), 5 mM MgCl₂).

FIGS. 24A-24B show the effect of backfilling on E-AB sensor performance.(24A) Gold electrodes were modified with 25 nM SCA-SW-40 and backfilledusing 3 mM dithiothreitol (DTT), 3 mM 6-mercapto-1-hexanol (MCH), or acombination of 1.5 mM DTT and 1.5 mM MCH. (24B) MDPV calibration curvesfor each electrode backfilled with either DTT, MCH or a combination ofDTT and MCH, with a measurement frequency of 150 Hz. Error bars shownare standard error for the mean of measurements using three separateelectrodes. Experiments were performed using the optimized E-AB sensorbuffer (10 mM Tris-HCl (pH 7.4), 5 mM MgCl₂).

FIGS. 25A-25C show E-AB detection using 5′-3′-tandem-truncated aptamer.(25A) Manual truncation of the 5′-overhang of SCA-SW-40 (SEQ ID NO: 18)to produce SCA-SW-34 (SEQ ID NO: 19). (25B) SCA-SW-34 is modified with a6-carbon linker and a thiol group at its 5′-end, and a methylene blueredox tag at its 3′-end, respectively. The immobilized aptamer remainsin a single-stranded flexible state in the absence of target,orientating the methylene blue redox tag away from the gold surface.(25C) In the presence of a synthetic cathinone drug, SCA-SW-34 undergoesa target-induced conformational change into a double-stranded structurewhich brings the redox tag close to the electrode surface, resulting inan increase in current.

FIGS. 26A-26B show the effect of 5′-overhang on E-AB sensor performance.(26A) Determination of aptamer surface coverage on gold electrodesmodified with either 25 or 50 nM of SCA-SW-34 or SCA-SW-40. (26B) Effectof frequency on signal gain using 30 MDPV with SCA-SW-34 or SCA-SW-40modified electrodes. Error bars shown are standard error for the mean ofmeasurements using three separate electrodes. Experiments were performedin selection buffer (10 mM Tris-HCl (pH 7.4), 20 mM NaCl, 0.5 mM MgCl₂).

FIGS. 27A-27B show the effect of 5′-overhang on E-AB sensor performance.(27A) Determination of aptamer surface coverage on gold electrodesmodified with either 15 or 25 nM of SCA-SW-34 or SCA-SW-40. (27B) Effectof frequency on signal gain using 30 MDPV with SCA-SW-34 or SCA-SW-40modified electrodes. Error bars shown are standard error for the mean ofmeasurements using three separate electrodes. Experiments were performedin selection buffer (10 mM Tris-HCl (pH 7.4), 20 mM NaCl, 0.5 mM MgCl₂).

FIGS. 28A-28B show sensitive MDPV detection using optimized conditions(10 mM Tris-HCl (pH 7.4), 0.1 mM NaCl, 0.03 mM MgCl₂). (A) E-AB sensorresponse to various concentrations of MDPV (0, 0.1, 0.5, 1, 5, 10, 25,50 and 100 μM). (B) Calibration curve of MDPV detection. Error barsshown are standard error for the mean of measurements using threeseparate electrodes.

FIG. 29 shows the cross-reactivity of the E-AB sensor against varioussynthetic cathinones at a concentration of 25 μM under optimized bufferconditions.

FIG. 30 shows the specificity of the E-AB sensor against variousinterferents at a concentration of 250 μM under optimized bufferconditions.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is the DNA sequence of a library contemplated for useaccording to the subject invention, where N is a random base.

SEQ ID NO: 2 is the sequence of a biotinylated cDNA contemplated for useaccording to the subject invention.

SEQ ID NO: 3 is the sequence of a forward primer contemplated for useaccording to the subject invention.

SEQ ID NO: 4 is the sequence of a biotinylated reverse primercontemplated for use according to the subject invention.

SEQ ID NO: 5 is the sequence of a reverse primer contemplated for useaccording to the subject invention.

SEQ ID NO: 6 is the DNA sequence of SCA2.1 contemplated for useaccording to the subject invention.

SEQ ID NOs: 7-17 are the DNA sequences of radomized regions ofcross-reactive aptamers contemplated for use according to the subjectinvention.

SEQ ID NO: 18 is the DNA sequence of SCA-SW-40 contemplated for useaccording to the subject invention.

SEQ ID NO: 19 is the DNA sequence of SCA-SW-34 contemplated for useaccording to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides a novel SELEX strategy for isolatingcross-reactive aptamers that recognize a core structure of asmall-molecule family and bind to structurally-similar molecules in saidfamily. The subject invention also provides methods, assays, andproducts for rapid, naked-eye detection of small molecules in a sample,in particular, in both clinical and field settings.

In one embodiment, the sample is a biological sample of a subject. Inspecific embodiments, the biological sample is selected from blood,plasma, urine, tears, sweat, and saliva. The subject may be any animalor human, preferably, a human. The subject may also be any animalincluding, but not limited to, non-human primates, rodents, dogs, cats,horses, cattle, pigs, sheep, goats, chickens, guinea pigs, hamsters andthe like.

In one embodiment, the sample is an environmental sample, for example,water, soil, air, or plant sample. In another embodiment, the sample isa seized drug sample, for instance, a street drug sample seized by lawenforcement or government officials.

Small Molecules

The term “target,” “small molecule,” or “small-molecule target,” as usedherein, includes any molecule capable of being detected using an aptamertechnique. In certain embodiments, the small molecule has a molecularweight less than 1000 Daltons, less than 900 Daltons, less than 800Daltons, less than 700 Daltons, less than 600 Daltons, less than 500Daltons, less than 400 Daltons, less than 300 Daltons, or less than 200Daltons.

In specific embodiments, the small-molecule target may be an amino acid,an amino acid-related molecule, a peptide, a steroid, a lipid, a sugar,a carbohydrate, a biomarker, a drug molecule, a drug metabolite, acoenzyme, a nucleotide (nt), a nucleotide-related molecule, a pyridinenucleotide, a cyclic nucleotide, or a cyclic dinucleotide. In otherembodiments, the small-molecule target may be an infective agent,antigen, toxin, disease biomarker, or a specific metal ion.

In one embodiment, the small molecule is a drug molecule. In oneembodiment, the drug molecule is a cathinone, a cathinone derivative, orsynthetic cathinone, such as a ring-substituted cathinone derivative orsynthetic cathinone. The synthetic cathinone has a general structure offormula (I)

wherein R¹ R², R³ and R⁴, are each independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, benzyl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,heterocycloalkyl, alkenyl, alkynyl, alkoxy, thiol, haloalkyl, acyl,halogen, amino, alkylamino, hydroxyl, hydroxylalkyl, and —COOH.

In one embodiment, R¹ is selected from the group consisting of hydrogen,alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl,alkenyl, alkynyl, haloalkyl, acyl, alkoxy, halogen, and hydroxylalkyl;R² is hydrogen or alkyl; and R³ and R⁴ are each independently selectedfrom the group consisting of hydrogen, alkyl, aryl, heteroaryl,cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl, alkynyl, haloalkyl,acyl, halogen, and hydroxylalkyl.

In a further embodiment, R¹ is halogen, such as fluorine, chlorine,bromine or iodine.

In some embodiments, R¹, taken together with the carbon atom to which itis attached and an adjacent carbon atom thereof, form a substituted orunsubstituted 5- or 6-membered homocyclic or heterocyclic ring. Forexample, R¹ may form a methylenedioxy group or aromatic ring such asbenzene with two adjacent carbon atoms of the ring where it is attached.

In other embodiments, R³ and R⁴, taken together with the nitrogen atomto which they are attached, form a substituted or unsubstituted 5- or6-membered heterocyclic ring. For example, R³ and R⁴ may form apyrrolidino group.

As used herein, “alkyl” means linear saturated monovalent radicals of atleast one carbon atom or a branched saturated monovalent of at leastthree carbon atoms. It may include hydrocarbon radicals of at least onecarbon atom, which may be linear. Examples include, but are not limitedto, methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, tert-butyl,pentyl, hexyl, and the like.

As used herein, “acyl” means a radical —C(O)R where R includes, but isnot limited to, hydrogen, alkyl or cycloalkyl, and heterocycloalkyl.Examples include, but are not limited to, formyl, acetyl, ethylcarbonyl,and the like. An aryl group may be substituted or unsubstituted.

As used herein, “alkylamino” means a radical —NHR or —NR2 where each Ris, independently, an alkyl group. Examples include, but are not limitedto, methylamino, (1-methylethyl)amino, dimethyl amino, methylethylamino,di(1-methylethyl)amino, and the like. An alkylamino may be substitutedor unsubstituted.

As used herein, “hydroxyalkyl” means an alkyl radical substituted withone or more hydroxy groups. Representative examples include, but are notlimited to, hydroxymethyl, 2-hydroxyethyl; 2-hydroxypropyl;3-hydroxypropyl; 1-(hydroxymethyl)-2-methylpropyl; 2-hydroxybutyl;3-hydroxybutyl; 4-hydroxybutyl; 2,3-dihydroxypropyl;2-hydroxy-1-hydroxymethylethyl; 2,3-dihydroxybutyl; 3,4-dihydroxybutyland 2-(hydroxymethyl)-3-hydroxy-propyl; preferably 2-hydroxyethyl;2,3-dihydroxypropyl and 1-(hydroxymethyl)-2-hydroxyethyl. A hydroxyalkylmay be substituted or unsubstituted.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 9 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₋₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from ethenyl; propen-1-yl; propen-2-yl;propen-3-yl; buten-1-yl; buten-2-yl; buten-3-yl; buten-4-yl;1-methyl-propen-1-yl; 2-methyl-propen-1-yl; 1-ethyl-ethen-1-yl;2-methyl-propen-3-yl; buta-1,3-dienyl; buta-1,2,-dienyl andbuta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no waylimited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and thelike.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain comprising one or more triple bonds. The alkynyl group may have 2to 9 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 4carbon atoms. The alkynyl group may be designated as “C₂₋₄ alkynyl” orsimilar designations. By way of example only, “C₂₋₄ alkynyl” indicatesthat there are two to four carbon atoms in the alkynyl chain, e.g., thealkynyl chain is selected from ethynyl, propyn-1-yl, propyn-2-yl,butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynylgroups include, but are in no way limited to, ethynyl, propynyl,butynyl, pentynyl, and hexynyl, and the like.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl.

As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclicor multicyclic aromatic ring system (including fused ring systems wheretwo carbocyclic rings share a chemical bond). The number of carbon atomsin an aryl group can vary. For example, the aryl group can be a C₆-C₁₄aryl group, a C₆-C₁₀ aryl group, or a C₆ aryl group. Examples of arylgroups include, but are not limited to, phenyl, benzyl, α-naphthyl,β-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl,biphenylenyl, and acenaphthenyl. Preferred aryl groups are phenyl andnaphthyl.

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcomprise(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

As used herein, “haloalkyl” refers to an alkyl group, in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include butare not limited to, chloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl, 1-chloro-2-fluoromethyl, and 2-fluoroisobutyl. Ahaloalkyl may be substituted or unsubstituted.

As used herein, a “substituted” group may be substituted with one ormore group(s) individually and independently selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, benzyl,substituted benzyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl,alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen,thiol, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato,isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl,haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, an amino,a mono-substituted amino group, a di-substituted amino group, andprotected derivatives thereof.

As used herein, “halogen” refers to an atom of fluorine, chlorine,bromine or iodine.

As used herein, “homocyclic ring” refers to cycloalkyl or aryl.

As used herein, “heterocyclic ring” refers to a ring, which may contain1 to 4 hetero-atoms selected from among nitrogen, oxygen, sulfur andother atoms in addition to carbon atoms.

Exemplary cathinones or synthetic cathinones include, but are notlimited to, 3, 4-methylenedioxypyrovalerone (MDPV);4′-methyl-α-pyrrolidinohexanophenone (MPHP); naphyrone; methylone;ethylone; butylone; pentylone; mephedrone; mexedrone; buphedrone;pentedrone; hexedrone; heptedrone; α-pyrrolidinopropiophenone (α-PPP);4′-methyl-α-pyrrolidinopropiophenone (M-α-PPP);3′,4′-methylenedioxy-a-pyrrolidinopropiophenone (MDPPP);1-phenyl-2-(1-pyrrolidinyl)-1-pentanone (α-PVP);α-pyrrolidinohexiophenone (α-PHP); α-pyrrolidinoheptiophenone (α-PHpP,PV8); diethylpropion; pyrovalerone; dimethylcathinone; diethylcathinone;methcathinone; ethcathinone; 3-methylmethcathinone (3-MMC);4-methylethcathinone (4-MEC); 3-chloromethcathinone (3-CMC);4-chloromethcathinone (4-CMC); n-ethyl-nor-pentedrone (NEP);3,4-methylenedioxy-a-pyrrolidinobutiophenone (MDPBP);4-methyl-α-pyrrolidinobutiophenone (MEPBP); 4-fluoromethcathinone(4-FMC); n-ethyl-nor-hexedrone (Hexen); n-ethyl-nor-heptedrone;4-ethylpentedrone; 4-methyl-NEP; and n-ethyl-nor-pentylone.

In a specific embodiment, the synthetic cathinone is MDPV, mephedrone,ethylone, naphyrone, penthylone, methylone, buthylone, MPHP, 4-MMC,methedrone, pyrovalenrone, MDPBP, α-PVP, MEPBP, 4-FMC, andmethcathinone.

Method of Identifying Cross-Reactive Aptamers

The subject invention provides a method of using a novel strategy toisolate and identify nucleic acid aptamers that cross-react with afamily of small molecules. The family of small molecules shares a corestructure. The aptamers identified and isolated by said method bind tothese structurally-similar compounds.

In one embodiment, the method employs a parallel-and-serial SELEXstrategy, comprising at least one step of a parallel selection and atleast one step of a serial selection. In another embodiment, the methodfor isolating an aptamer for a family of structurally-similar smallmolecules comprises: providing a nucleic acid library, mixing the smallmolecules in said family with the nucleic acid library, binding thesmall molecules to one or more aptamers in the nucleic acid library,separating the aptamer bound to the small molecules in said family fromat least a portion of the unbound nucleic acid molecules, and isolatingthe aptamer and optionally, amplifying the isolated aptamer.

In one embodiment, the method for isolating a cross-reactive aptamercomprises the steps of:

-   -   providing a nucleic acid library;    -   selecting two or more targets from a target family having a core        structure to be recognized by the aptamer;    -   performing at least one round of a parallel selection against        the selected two or more targets in the target family;    -   collecting pools of aptamers for each of the selected two or        more targets;    -   performing at least one cycle of a serial selection against the        selected targets in the target family in a sequential order; and    -   collecting the cross-reactive aptamer that binds to the selected        two or more targets of the target family.

In one embodiment, the target family comprises as many targets as neededthat have variations at all desired substituent sites while having thesame core molecular framework. The target selection defines the corestructure to be recognized by the isolated aptamer, and createsselection pressure for isolating aptamers that are insensitive to theidentity of peripheral substituents.

In one embodiment, the target family is synthetic cathinones, a familyof designer drugs that share the same beta-keto phenethylamine corestructure.

In one embodiment, the nucleic acid library is a DNA library.

As used herein, the terms “nucleic acid library,” “polynucleotidelibrary,” or the like, generally refer to a mixture of nucleic acidmolecules having variable sequence from which an aptamer is selected fora specific target family of small molecules. The oligonucleotidesmolecules of the library have a length ranging from about 5 to about 500bases, or to about 450 bases, or to about 400 bases, or to about 350bases, or to about 300 bases, or to about 250 bases, or to about 200bases, or to about 150 bases, or to about 100 bases, or to about 50bases. In some embodiments, the oligonucleotides molecules of thelibrary have a length between about 10 bases and about 100 bases, orbetween about 20 bases and about 90 bases, or between about 30 bases andabout 70 bases, or between about 40 bases and about 60 bases. In certainembodiments, the oligonucleotides molecules of the library have a lengthof 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, or 80 bases.

The constituent molecules of a nucleic acid library may be naturallyoccurring nucleic acids or fragments thereof (e.g., in a cDNA or ESTlibrary), chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made using any combination ofthe aforementioned techniques. In some embodiments, each nucleic acidmolecule in the library may include one or more fixed (e.g., known)nucleotide sequences 5° to, 3° to, or flanking, the variable region forthe purpose of facilitating the enrichment and identification of targetaptamers (such as by using PCR, affinity chromatography, or any similarmethods used to purify or enrich target nucleic acids).

In one embodiment, the library pool is challenged with at least two, atleast three, at least four, at least five, or at least six differenttargets sharing the same core structure in each parallel selectionround, respectively, and then sequentially in a serial selection cycleto isolate an aptamer that can cross-react to at least two, at leastthree, at least four, at least five, or at least six different targets,and ideally all target-analogs sharing the same core structure.Advantageously, challenging the library pool with more targets from thesmall-molecule family results in aptamers with broader target-bindingspectra and higher cross-reactivity.

In the step of parallel selection, multiple aptamer pools are enrichedusing each individual target from the target family. Cross-reactiveaptamers recognizing the shared core structure are enriched in each ofthese pools, while aptamers specific to an individual target from thetarget family are only enriched in their respective pool. Therefore,when all pools are combined after a few rounds of parallel selection,cross-reactive aptamers are highly enriched. The combined pool is thensubjected to serial selection with each target molecule sequentially, aprocess that ultimately retains only those aptamers that bind to thecore structure shared by these targets.

In one embodiment, the method uses SELEX strategy for isolating across-reactive aptamer for synthetic cathinones, said method comprisesthe steps of:

-   -   providing a DNA library;    -   selecting at least three synthetic cathinone targets;    -   performing at least one round of a parallel selection against        each selected synthetic cathinone target to isolate a pool of        aptamers for each selected synthetic cathinone target;    -   collecting the isolated pools of aptamers for each selected        synthetic cathinone target;    -   combining the isolated pools of aptamers for each selected        synthetic cathinone target;    -   performing at least one cycle of a serial selection against each        selected synthetic cathinone target in each round sequentially;        and    -   collecting the cross-reactive aptamer.

In one embodiment, the DNA library comprises more than one librarypools. The same or different library pools may be used for each of thesynthetic cathinones in the family. In a specific embodiment, the DNAlibrary comprises 6×10¹⁴ oligonucleotides. Each library strand isstem-loop structured and has 73 nucleotides in length, with an8-base-pair stem and a randomized 30 nucleotide loop.

In a preferred embodiment, the DNA library comprises a sequence of SEQID NO: 1 where N represents a random base and N30 represents therandomized 30 nucleotides. The randomized nucleotides are eachindependently selected from adenine (A), thymine (T), cytosine (C), andguanine (G). Preferably, the randomized region has a sequence selectedfrom SEQ ID NOs: 7-17.

In one embodiment, the cross-reactive aptamer isolated by the methodaccording to the subject invention is capable of binding to syntheticcathinones that share the same core structure.

In a further embodiment, the synthetic cathinone family includes, butnot limited to, α-PVP, ethylone, butylone, pyrovalerone, MPHP, 4-MMC,MDPV, methedrone, naphryone, MDPBP, 3-FMC, 4-FMC, pentylone,methcathinone, methylone, and MEPBP.

In a specific embodiment, the method uses three targets, α-PVP,ethylone, and butylone from the target synthetic cathinone family forboth parallel selection and serial selection. Parallel selection isperformed using three different initial library pools, with one poolbeing challenged with each of α-PVP, ethylone and butylone. In someembodiments, the parallel selection is performed for at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 rounds. Ineach round, the targets may be used at same or different concentrations.Preferably, the targets may be used in a round of the parallel selectionat a lower concentration than that used in the previous round becausereducing target concentration round-by-round increases selectionstringency.

In one embodiment, the method further comprises performing counter-SELEXagainst at least one structurally-similar non-cathinone molecule in eachround of the parallel selection, preferably, prior to the positiveselection. The structurally-similar non-cathinone molecules include, butare not limited to, acetaminophen, amphetamine, cocaine, ephedrine,lidocaine, methamphetamine, procaine, promazine, and pseudoephedrine.Counter-SELEX against structurally-similar non-cathinone moleculeseliminates the non-specific binding aptamers and ensures that theselected and enriched aptamer has high specificity to syntheticcathinones. In some embodiments, the number of counter targets and theconcentrations of counter targets are progressively increased during theselection process to increase selection stringency.

In specific embodiments, the parallel selection is performed for fiverounds. During the first round of the parallel selection, each initiallibrary pool is challenged with a high concentration of targets (e.g.,1000 μM), and eluted strands are collected and amplified by PCR for thenext round of selection. In the beginning of the second round,counter-SELEX is performed against structurally-similar non-cathinonemolecules. In round two, counter-SELEX is first performed for each poolagainst cocaine (e.g., 100 μM) with positive selection then performedwith a lower concentration of target (e.g., 500 μM). In the third round,the same target concentration is used but an additional counter-target(e.g., 100 μM procaine) is included. In rounds four and five,counter-SELEX is performed against cocaine, procaine, and lidocaine eachat a concentration of, for example, 100 μM in a mixture with a furtherlowered concentration of target (e.g., 250 μM) for positive selection.After the fifth round, a gel elution assay is utilized to determine thetarget-binding affinity of each pool to their respective target.

In one embodiment, the serial selection is performed to enrichcross-reactive aptamers and exclude aptamers only specific to individualtarget. The serial selection may be performed more than one cycle, inwhich the aptamer library is selected against each target of thecompound family in a sequential order. Specifically, the serialselection comprises the steps of:

-   -   providing an aptamer library that is obtained by combining each        pool obtained from the parallel selection,    -   challenging the aptamer library with targets of the synthetic        cathinone family sequentially for at least one cycle with one        target being used in each round within one cycle, and    -   collecting a pool of aptamers, the pool consisting of        cross-reactive aptamers binding to synthetic cathinones that        share the same core structure.

In one embodiment, the serial selection further comprises performingcounter-SELEX against at least one structurally-similar non-cathinonemolecule in each round of the challenge prior to the positive selectionagainst the target molecule in each cycle of the serial selection. Thestructurally-similar non-cathinone molecules include, but are notlimited to, acetaminophen, amphetamine, cocaine, ephedrine, lidocaine,methamphetamine, procaine, promazine, and pseudoephedrine.

In one embodiment, the method for isolating a cross-reactive aptamer forsynthetic cathinones comprises the steps of:

-   -   providing a aptamer library, the library being SEQ ID NO: 1;    -   performing at least one round of parallel selection against        α-PVP, ethylone, and butylone, respectively;    -   collecting three pools of aptamers, each pool being specific to        α-PVP, ethylone, or butylone;    -   obtaining a combined pool of aptamer by combining the aptamer        pools each specific to α-PVP, ethylone, and butylone;    -   performing at least one cycle of serial selection against α-PVP,        ethylone, and butylone in each round in a sequential order; and    -   collecting the cross-reactive aptamer for synthetic cathinones.

In a specific embodiment, the serial selection comprises i) challengingthe combined pool against butylone; ii) collecting a first pool ofaptamer that binds to butylone; iii) challenging the first pool ofaptamer of step ii) against ethylone; iv) collecting a second pool ofaptamer, the second pool of aptamer being able to bind to both butyloneand ethylone; v) challenging the second pool of aptamer of step iv)against α-PVP; and vi) collecting a third pool of aptamer, the thirdpool of aptamer being able to bind to all three targets: butylone,ethylone and α-PVP. Advantageously, the third pool of aptamer is thecross-reactive aptamer that binds to other synthetic cathinones havingthe same core structure as butylone, ethylone and α-PVP.

In one embodiment, the method further comprises evaluating thecross-reactivity and specificity of the resulting pool after each round,each cycle, and each selection using, for example, gel elution assay.The pool affinity towards each target of the tested family increases ineach round and/or cycle of the serial selection.

In some embodiments, the method further comprises amplifying theresulting pool of aptamers through PCR prior to the nextround/cycle/selection. The method further comprises purifying thecross-reactive aptamer from the final enriched pool and amplifying thepurified cross-reactive aptamer.

Importantly, the parallel-and-serial selection strategy is designed topromote the enrichment of aptamers that identify the core structureshared by a given target family while being tolerant to side-chainsubstituents. Rational target selection defines the targeted corestructure, and the use of parallel and serial selection forces theisolation of desired cross-reactive aptamers. The strategy describedherein can be used to isolate cross-reactive aptamers for other familiesand classes of structurally-related small molecules in applications suchas medical diagnostics, environmental monitoring, food safety, andforensic science.

Cross-Reactive Aptamers

In one embodiment, the cross-reactive aptamers are identified using theparallel-and-serial selection strategy according to the subjectinvention. The cross-reactive aptamers are capable of binding to a corestructure of structurally-similar compounds in a family of interest.

In one embodiment, the cross-reactive aptamer is an oligonucleotide,such as DNA or RNA molecules and may be single-stranded ordouble-stranded. In a preferred embodiment, the aptamer is a DNAaptamer.

The cross-reactive aptamer may be partially or fully folded to formvarious secondary structures (e.g., stems, loops, bulges, pseudoknots,G-quadruplexes and kissing hairpins), which in turn can form uniquethree-dimensional architectures able to specifically recognize theirtargets by exploiting a variety of interactions—such as hydrophobic andelectrostatic interactions, hydrogen bonding, van der Waals forces, andπ-π stacking as well as shape complementarity.

As used herein, the terms “polynucleotide,” “nucleotide,”“oligonucleotide,” and “nucleic acid” refer to a nucleic acid comprisingDNA, RNA, derivatives thereof, or combinations thereof.

In certain embodiments, the cross-reactive aptamer according to thepresent invention may comprise at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, or at least 80 nucleotides. Thecross-reactive aptamer, preferably, comprises 20 to 200 nucleotides,preferably 25 to 150 nucleotides, more preferably 30 to 100 nucleotides,most preferably, 35 to 60 nucleotides.

In certain embodiments, the cross-reactive aptamer according to thepresent invention has a minimum length of, for example, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40nucleotides. The aptamer according to the present invention may have amaximum length of, for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or120 nucleotides. The aptamer according to the present invention may havea length of, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides.

In some embodiments, the cross-reactive aptamers have free ends. Forexample, the 3′ and 5′ ends may not be ligated to form a loop, althoughthey may be conjugated to other molecules or otherwise modified. Theaptamers may adopt a tertiary structure such as a hairpin loop.

In certain embodiments, the cross-reactive aptamer comprises at leastone stem, two stems, or three stems. Each stem may be fully or partiallycomplementary. Each stem may comprise the same or a different number ofnucleotides. Exemplary lengths of each stem may be 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 base pairs. A partially complementary stemmay comprise more than one wobble base pair, including, but not limitedto, G-U, and T-G.

In one embodiment, the cross-reactive aptamer comprises at least onejunction, which is formed when two or more stems meet. In certainembodiments, the junction may be a loop between two stems, or athree-way junction (TWJ). The junction in an aptamer can serve as abinding domain for a small-molecule target.

In one embodiment, the cross-reactive aptamer has at least onehairpin/stem-loop structure. The loop may have a minimum length of, forexample, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The loop may havea maximum length of, for example, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. The loopmay comprise, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In a specificembodiment, the loop comprises 28 nucleotides. The loop region is thetarget-binding site of the aptamer.

In a specific embodiment, the cross-reactive aptamer recognizes the corestructure of synthetic cathinones and binds to all synthetic cathinones.The synthetic cathinones include, but are not limited to MDPV, ethylone,naphyrone, penthylone, methylone, buthylone, MPHP, 4-MMC, methedrone,pyrovalenrone, MDPBP, α-PVP, MEPBP, 4-FMC, and methcathinone.

In specific embodiments, the cross-reactive aptamers selected using theparallel-and-serial selection strategy are synthetic-cathinone-bindingaptamers. In a further embodiment, the cross-reactive aptamer is a DNAaptamer comprising 46 nucleotides. In some embodiments, thecross-reactive aptamer comprises a stem and a loop region, the loopregion having a sequence selected from SEQ ID NOs: 7-17.

In a preferred embodiment, the cross-reactive aptamer is SCA2.1 (SEQ IDNO: 6) or sequences sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 99% identity with SCA2.1 (SEQ ID NO: 6). SCA2.1 may befolded as a stem-loop structure or may comprise two hairpin structures.The structure of SCA2.1 may vary when it is present in buffers withdifferent salt concentrations. In a specific embodiment, SCA2.1comprises a 9-base-pair stem and a 28-nucleotide loop.

SCA2.1 recognizes the beta-keto phenethylamine core structure ofsynthetic cathinones and binds to synthetic cathinones. Variations inthe side chains do not significantly affect target-binding affinity.SCA2.1 does not cross-react with interferents such as pseudoephedrine,promazine, procaine, ephedrine, acetaminophen, methamphetamine,lidocaine, amphetamine, cocaine, sucrose, and caffeine.

The cross-reactive aptamer of the present invention may be chemicalmodified. The chemical modifications as described herein include achemical substitution at a sugar position, a phosphate position, and/ora base position of the nucleic acid including, for example,incorporation of a modified nucleotide, incorporation of a cappingmoiety (e.g., 3′ capping), conjugation to a high molecular weight,non-immunogenic compound (e.g., polyethylene glycol (PEG)), conjugationto a lipophilic compound, and substitutions in the phosphate backbone.Base modifications may include 5-position pyrimidine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo- or 5-iodo-uracil, and backbone modifications.Sugar modifications may include 2′-amine nucleotides (2′-NH2).2′-fluoronucleotides (2′-F), and 2′-O-methyl (2′-OMe) nucleotides. Suchmodifications may improve the stability of the aptamers or make theaptamers more resistant to degradation. In some embodiments, each baseof a given type (e.g., A, T, C, and G) may contain the same chemicalmodification.

The cross-reactive aptamers may be modified by addition of one or morereporter labels (or detectable labels). In some embodiments, the labelmay be attached to either the 5′ or 3′ end of the aptamer. The label mayalso be attached to the backbone of the aptamer. The skilled person willbe aware of techniques for attaching labels to nucleic acid strands. Thedetectable label may be attached directly or indirectly to the nucleicacid aptamer. If the label is indirectly attached to the nucleic acidaptamer, it may be by any mechanism known to one of skill in the art,such as using biotin and streptavidin.

The cross-reactive aptamers may comprise a reporter label, such as afluorescent dye, nanoparticle, or an enzyme. Exemplary labels include,but are not limited to, an organic donor fluorophore or an organicacceptor fluorophore, a luminescent lanthanide, a fluorescent orluminescent nanoparticle, an affinity tag such as biotin, or apolypeptide. In some embodiments, the aptamer may comprise a fluorescentlabel, for example, fluorescein, TAMRA, rhodamine, Texas Red, AlexaFluor (e.g., AlexaFluor 488, AlexaFluor 532, AlexaFluor 546, AlexaFluor594, AlexaFluor 633 and AlexaFluor 647), cyanine dye (e.g.,diethylthiatricarbocyanine (Cy7), Cy7.5, Cy5, Cy5.5 and Cy3), Tye dye(e.g., TYE 563, TYE 665, TYE 705), atto dye (e.g., Atto 594 and Atto633), Hexachlorofluorescein, FAM (6-carboxyfluroescein), BODIPY FL,OliGreen, 40,6-diamidino-2-phenylindol (DAPI), Hoechst 33,258, malachitegreen (MG), and FITC. The nanoparticle can be an upconversionnanoparticle. In some embodiments, the fluorophore is selected from thegroup consisting of fluorophores that emit a blue, green, near red orfar red fluorescence.

In one embodiment, the reporter label is a fluorescent dye and quencherpair. The quenchers can be, for example, Dabcyl, DDQ-I, Eclipse, IowaBlack FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, or BHQ-3.

In one embodiment, the reporter label can be an electroactive molecule,for example, methylene blue, ferrocene, or all enzymes that can convertnonelectroactive substrates into electroactive products.

In one embodiment, the cross-reactive aptamer can bind to a dye such asa fluorophore within the target binding domain or the loop domain. In apreferred embodiment, the dye is diethylthiatricarbocyanine (Cy7).

In one embodiment, the cross-reactive aptamer has inherentdye-displacement functionality. A dye can bind within the target bindingdomain or the loop domain of the cross-reactive aptamer, and can bedisplaced in the presence of a small-molecule target, resulting in achange in absorbance, color, or fluorescence. Such change may occurwithin seconds. Such change can also directly reflect the extent oftarget binding and be used for detection and quantitative measurement ofthe target concentration.

In one embodiment, the cross-reactive aptamer binds to thesmall-molecule target with a dissociation constant of, for example,about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM,about 7 μM, about 8 μM, about 9 μM, or about 10 μM. In specificexamples, the aptamer binds to the small molecule with a dissociationconstant between about 0.001 μM and about 1000 μM, between about 0.01 μMand about 500 μM, between about 0.1 μM and about 200 μM, between about0.5 μM and about 100 μM, between about 1 μM and about 100 μM, betweenabout 1 μM and about 50 μM, between about 1 μM and about 30 μM, betweenabout 1 μM and 20 μM, or between about 1 μM and about 10 μM.

Method of Using the Cross-Reactive Aptamer

The subject invention provides aptamer-based sensors for rapid andnaked-eye detection of small-molecule targets. The aptamer-based sensorcomprises a cross-reactive aptamer that binds to a core structure ofstructurally-similar compounds in a family of interest. The subjectinvention also provides methods of using the aptamer-based sensor fordetecting a family of small-molecule targets in a complex sample.

In one embodiment, the subject invention provides an assay employingdye-displacement strategies for the detection of small-molecule targets.In such assay, a small-molecule dye is initially associated with thebinding domain of an aptamer. The presence of the small-molecule targetscauses displacement of the dye from the binding domain, resulting in anabsorbance, color, or fluorescence change.

In one embodiment, the aptamer according to the subject invention doesnot require any additional labeling or chemical modification. Themethods for detecting small molecule targets using such aptamer withoutany labeling are label-free.

In one embodiment, the subject invention provides a method for rapid andsensitive detection of a family of small-molecule targets in a samplecomprising contacting the sample with a aptamer-based sensor selectivefor the family of small-molecule targets, wherein the aptamer-basedsensor comprises a cross-reactive aptamer that binds to the corestructure of the family of small-molecule targets and a dye, anddetecting the small-molecule target in the sample, wherein the detectionof the small-molecule target comprises measuring a signal generated uponbinding of the small-molecule targets to the loop domain of the aptamer.

In a specific embodiment, the dye is Cy7. Cy7 is a small-molecule dyethat exists in equilibrium between monomer and dimer forms, which hasabsorbance peaks at 760 and 670 nm, respectively. Cy7 monomer can bindinto hydrophobic target-binding domains of aptamers, which results in asignificant enhancement of absorbance at 760 nm. The binding of targetto the aptamer can displace Cy7 monomer from the binding domain withinseconds, which causes the dye to dimerize in aqueous solution. Thisresults in the reduction of absorbance at 760 nm and enhancement ofabsorbance at 670 nm, which enables Cy7 to be used as a colorimetricindicator for small molecule detection. Also, the signal change may beindicated using the absorbance ratio at 670/760 nm.

In another embodiment, the method further comprises determining theconcentration of the small-molecule target in the sample. Thedetermination can comprise comparing the signal generated upon targetbinding with a standard curve of such signal. For example, thedetermination based on Cy7 displacement assay comprises comparing theabsorbance signal generated upon binding of aptamer-target complex witha standard curve of the absorbance of Cy7, or a standard curve of theabsorbance ratio at 670/760 nm. The absorbance read-out can bequantified in seconds by, for example, a microplate-reader or portablephotometer, allowing for high-throughput or on-site detection,respectively.

In one embodiment, the cross-reactive aptamer is asynthetic-cathinone-binding aptamer that specifically binds to thesynthetic cathinones having the same beta-keto phenethylamine corestructure. Preferably, the synthetic-cathinone-binding aptamer has asequence of SEQ ID NO: 6.

In one embodiment, the synthetic-cathinone-binding aptamer also binds toCy7 in its target-binding site in the loop region. Thus, thesynthetic-cathinone-binding aptamer can be used in Cy7 displacementassay for detecting one or more synthetic cathinones in a sample. Withincreased concentration of aptamer, a gradual absorbance peak shift from760 to 775 nm occurs, which shows that absorbance of the monomer canchange in different microenvironments, such as when the dye binds to theaptamer. Specifically, Cy7 has a K_(D) of 1.6 μM at 775 nm. In someembodiment, the signal change may be indicated using the absorbanceratio at 670/775 nm.

In one embodiment, the method for rapid, sensitive and naked-eyedetection of synthetic cathinones in a sample comprises contacting thesample with a aptamer-based sensor selective for synthetic cathinones,wherein the aptamer-based sensor comprises a cross-reactive aptamer thatbinds to synthetic cathinones and Cy7, and detecting whether a change incolor occurs, the change in color being indicative of the presence ofthe synthetic cathinones in the sample.

In such assay, Cy7 is initially associated with the binding domain ofthe synthetic-cathinone-binding aptamer. The presence of syntheticcathinones causes the displacement of Cy7 from the binding domain,resulting in a reduction of absorbance at 775 nm and enhancement ofabsorbance at 670 nm, resulting in a color change from colorless tobright blue that can be detected by naked eyes.

In some embodiments, the dye, e.g., Cy7 may be used at a concentrationranging from about 1 μM to about 50 μM, from about 1 μM to about 40 μM,from about 1 μM to about 30 μM, from about 1 μM to about 20 μM, fromabout 1 μM to about 10 μM, from about 1 μM to about 9 μM, from about 1μM to about 8 μM, from about 1 μM to about 7 μM, from about 1 μM toabout 6 μM, from about 2 μM to about 6 μM, about 2.2 μM to about 6 μM,about 2.5 μM to about 6 μM, about 3 μM to about 6 μM, and from about 3.5μM to about 5 μM. In certain embodiments, the dye, e.g., Cy7 is used ata concentration of 2.5 μM, 2.8 μM, 3 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM,3.6 μM, 3.7 μM, 3.8 μM, 3.9 μM, 4 μM, 4.2 μM, 4.5 μM, 4.8 μM or 5 μM. Ina preferred embodiment, Cy7 is used at a concentration of 3.5 μM.

Advantageously, the assay has excellent specificity because the aptamerdoes not cross-react to non-synthetic cathinone interferents. Also, thecolorimetric Cy7-displacement assay can detect nanomolar syntheticcathinone concentrations, even in urine and saliva in a label-freemanner via instrumental means. This colorimetric Cy7-displacement assaycan also achieve instantaneous detection of as low as 6.3 μM target(e.g., ethylone) with the naked-eye when Cy7 is used at a micromolarconcentration.

The method of the subject invention is remarkably simple, fast andspecific. For example, the detection can be performed in a single tubecontaining the aptamer-based sensor and the sample of interest. Notably,this method is more cross-reactive to synthetic cathinones than anyexisting immunoassays while being more cost-effective (30 cent/sample)and having a detection limit suitable for screening of these drugs inbiosamples.

The methods of the subject invention can be label-free and, in certainembodiments, can detect synthetic cathinones with the naked eye withinseconds. Because the color intensity of the solution is proportional tothe concentration of synthetic cathinones, the method of the subjectinvention can be used to determine the concentration of syntheticcathinones in the sample.

In one embodiment, the method according to the subject invention allowsfor the visual detection of a variety of synthetic cathinones including,but are not limited to, α-PVP, ethylone, buthylone, naphyrone, MDPV,pentylone, methylone, 4-MMC, 4-FMC, cathinone, 3-FMC, and methcathinone.

In one embodiment, the subject invention provides a method forgenerating cross-reactive aptamers with structure-switchingfunctionality and a means of rapid and sensitive detection of asmall-molecule target family. The generation of a structure-switchingcross-reactive aptamer entails digesting the cross-reactive aptamer withan exonuclease mixture, such as Exo III and Exo I.

In one embodiment, the cross-reactive aptamer is truncated from the 3′and/or 5′ end by nucleases, such as Exo III and Exo I that exhibit3′-to-5′ exonuclease activity on double-stranded and single-strandedDNA, respectively. Exo III is sensitive to local structural changes indouble-stranded DNA induced by small-molecule binding. Exo III catalyzes3′-to-5′ digestion of aptamers by a stepwise removal of mononucleotides,forming single-stranded products. Exo I specifically digestssingle-stranded nontarget-bound unfolded aptamers, but is resistant totarget-bound folded aptamers. A combined Exo III and Exo I digestiongenerates truncated aptamers with structure-switching functionality. Incertain embodiments, such truncation or digestion of an aptamer may bestopped at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides orbase pairs prior to the target-binding site. Such truncated or digestedaptamer product is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12nucleotides or base pairs shorter compared to the pre-folded aptamerbefore the truncation or digestion by the exonuclease mixture.

The resulting digestion product has structure-switching functionalitywith similar or equal affinity as its parent cross-reactive aptamer. Inthe absence of the target, the truncated aptamer exists in an unfoldedsingle-stranded state while in the presence of the target, the truncatedaptamer folds into a double-strand structure with the target binding inthe binding domain. Such structure-switching cross-reactive aptamer canbe directly employed in folding-based aptamer sensors.

In specific embodiments, the truncated or digested aptamer has a stemcomprising a sticky end or a blunt end. The truncated aptamer having asticky end comprises a 5′ or 3′ overhang. 5′-3′-tandem-truncated aptamerhas a stem comprising a blunt end.

In a specific embodiment, the structure-switching cross-reactive aptameris SCA-SW-40 (SEQ ID NO: 18), SCA-SW-34 (SEQ ID NO: 19) or sequencessharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%identity with SCA-SW-40 (SEQ ID NO: 18) or SCA-SW-34 (SEQ ID NO: 19).SCA-SW-40 (SEQ ID NO: 18) comprises nucleotides 1-40 of SEQ ID NO: 6while SCA-SW-34 (SEQ ID NO: 19) comprises nucleotides 7-40 of SEQ ID NO:6.

In one embodiment, the subject invention provides a method for detectinga small molecule target or a small-molecule target family in a sampleusing the structure-switching cross-reactive aptamer.

In one embodiment, the subject invention provides methods for rapid andsensitive detection of a small-molecule target or a small-moleculetarget family in a sample by incorporating the structure-switchingcross-reactive aptamers into an electrochemical aptamer-based (E-AB)sensor, which has demonstrated target-induced conformational changeswithin the aptamers and has achieved excellent sensor performance. Themethod comprises contacting the sample with the E-AB sensor, anddetecting the small-molecule target/target family in the sample, whereinthe detection of the small-molecule target comprises measuring a signalgenerated from a signal reporter.

In one embodiment, the E-AB sensor comprises a structure-switchingcross-reactive aptamer, and an electrode, wherein thestructure-switching cross-reactive aptamer is labeled with a redox tagat one end and a functional group at the other end, and wherein thestructure-switching cross-reactive aptamer is conjugated to the surfaceof the electrode via the functional group. The functional groupsinclude, but are not limited to, thiol, sulfide, disulfide, amide,ester, alkenyl, alkynyl, carbonyl, aldehyde, carboxylate, carboxyl, andcarbonate ester groups. Preferably, the functional group is thiol, andthe redox tag is a methylene blue (MB) redox tag, which may label theaptamer at the 5′ end, and 3′ end via a linker having 1-10 carbons,preferably, a linker having 6 or 7 carbons.

In the absence of a target, the structure-switching cross-reactiveaptamer is primarily unfolded, prohibiting electron transfer from theredox tag to the electrode. In the presence of a target, thestructure-switching cross-reactive aptamer undergoes a target-inducedconformational change that brings the redox tag close to the electrodesurface, facilitating efficient electron transfer and resulting in anincrease in current within seconds. In a further embodiment, thedetection of the small-molecule target comprises measuring a signalgenerated upon binding of the small-molecule target with the E-ABsensor, wherein the signal is an increase in current.

In one embodiment, the electrode is made of, for example, gold, silver,or platinum. The electrode may have any shape suitable for the E-ABsensor. Exemplary shapes of electrode include, but are not limited to,rod, sheet, plate, and disc. The electrode may have a size ranging fromabout 100 nm to about 50 mm, from about 500 nm to about 10 mm, fromabout 1 mm to about 5 mm. In a specific embodiment, the electrode has adiameter about 3 mm.

In certain embodiments, the aptamer is immobilized on the electrode ofthe E-AB sensor at a density ranging from about 1×10¹⁰ to about 1×10¹⁵,about 5×10¹⁰ to about 5×10¹⁴, about 1×10¹¹ to about 1×10¹⁴, about 5×10¹¹to about 5×10¹³, about 1×10¹² to about 5×10¹³, and about 1×10¹² to about1×10¹³ molecules/cm². In specific embodiment, the aptamer is immobilizedon the electrode at a density of 0.73×10¹², 1.02×10¹², 1.5×10¹²,1.57×10¹², 3×10¹², 3.2×10¹², 7.3×10¹², or 12×10¹² molecules/cm².

In one embodiment, the E-AB sensor further comprises a backfiller tofill vacant areas on the electrode surface. The backfiller includes DTT,MCH, and/or a combination thereof. The backfiller is immobilized on thesurface of the electrode, for example, via thiol-gold chemistry.

In certain embodiments, the backfiller may be used at concentrationsbetween about 10 μM to about 50 mM, from about 100 μM to about 40 mM,from about 200 μM to about 30 mM, from about 500 μM to about 20 mM, fromabout 1 mM to about 10 mM, from about 2 mM to about 9 mM, and from about2 mM to about 5 mM. In a preferred embodiment, the backfiller is used,either alone or in combination, at a concentration of 1 mM, 2 mM, 3 mM,4 mM, or 5 mM.

In a specific embodiment, the E-AB sensor comprises astructure-switching cross-reactive aptamer, and an electrode, whereinthe electrode is a gold electrode, wherein the structure-switchingcross-reactive aptamer is labeled with a 5′ thiol and a 3′ methyleneblue (MB) redox tag, and conjugated to a gold electrode surface viathiol-gold chemistry.

In one embodiment, the method further comprises determining theconcentration of the small-molecule target in the sample. Thedetermination comprises comparing the current generated upon binding ofthe small-molecule target with the E-AB sensor with a standard curve.The read-out can be quantified in seconds by, for example, a voltmeteror a potentiostat. Thus, the current measured upon binding of thesmall-molecule target with the E-AB sensor is indicative of the presenceof the small-molecule target in such sample.

In one embodiment, the method according to the subject invention can beused to detect one or more synthetic cathinones in a sample. The methodcomprises contacting the sample with an E-AB sensor, wherein the E-ABsensor comprises a structure-switching cross-reactive aptamer selectivefor synthetic cathinones and the structure-switching cross-reactiveaptamer is conjugated to the surface of a gold electrode; and detectingone or more synthetic cathinones in the sample, wherein the detectioncomprises measuring a current generated upon binding of syntheticcathinones with the E-AB sensor. Advantageously, this method using E-ABsensor can detect synthetic cathinones in a sample within 10 seconds ofthe reaction.

In one embodiment, the E-AB sensor is used to detect syntheticcathinones in a buffer solution comprising at least one salt containing,for example, Mg²⁺ and/or Na⁺. The salt may be, for example, MgCl₂ and/orNaCl. The salt may be used at the physiological concentration or anyconcentrations suitable for maintaining the function and bindingaffinity of isolated aptamers and the E-AB sensor.

Exemplary concentrations of magnesium salt may be between about 0 mM andabout 50 mM, between about 0.1 mM and about 40 mM, between about 0.2 mMand about 30 mM, between about 0.5 mM and about 20 mM, between about 1mM and about 15 mM, between about 2 mM and about 10 mM, between about 3mM and about 8 mM, between about 0 mM and about 5 mM, between about 0 mMand about 2 mM, between about 0 mM and about 1 mM, between about 0.01 mMand about 0.5 mM, and between about 0.02 mM and about 0.1 mM. In aspecific embodiment, the concentration of magnesium salt is 0.03 mM or 5mM.

Exemplary concentrations of sodium salt may be between about 0 mM andabout 50 mM, between about 0 mM and about 40 mM, between about 0 mM andabout 30 mM, between about 0 mM and about 20 mM, between about 0 mM andabout 15 mM, between about 0 mM and about 10 mM, between about 0 mM andabout 5 mM, between about 0 mM and about 1 mM, between about 0.05 mM andabout 1 mM, and between about 0.1 mM and about 0.5 mM. In a specificembodiment, the concentration of sodium salt is 0 or 0.1 mM.

In a specific embodiment, the structure-switching cross-reactive aptamer(e.g., SCA-SW-40 or SCA-SW-34) is modified with a 5′ thiol and a 3′methylene blue redox tag. The thiol group may be linked to the 5′ end ofthe aptamer via a first linker and the methylene blue redox tag may belinked to the 3′ end of the aptamer via a second linker. The first andsecond linkers may be different or identical. Each of the first andsecond linkers independently comprises 1-10 carbons. Preferably, each ofthe first and second linkers independently comprises 2-8 carbons. Morepreferably, the first linker is a 6-carbon linker (i.e., —(CH₂)₆—) andthe second linker is a 7-carbon linker (i.e., —(CH₂)₇—).

In some embodiments, the aptamer according to the subject invention maybe used at a concentration ranging from about 1 nM to about 10 mM, about10 nM to about 5 mM, about 20 nM to about 2 mM, about 50 nM to about 1mM, about 100 nM to about 500 μM, about 200 nM to about 200 μM, about500 nM to about 100 μM, about 1 μM to about 50 μM, from about 1 μM toabout 40 μM, from about 1 μM to about 30 μM, from about 1 μM to about 20μM, from about 1 μM to about 10 μM, from about 2 μM to about 9 μM, fromabout 2 μM to about 8 μM, from about 2 μM to about 7 μM, from about 3 μMto about 6 μM, from about 4 μM to about 6 μM, and from about 5 μM toabout 6 μM. In specific embodiments, the aptamer according to thesubject invention may be used at a concentration of 1 nM, 10 nM, 20 nM,25 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 3 μM, 4 or 5 μM.

In one embodiment, the method according to the subject invention canachieve superior sensitivity for target detection at low micromolar ornanomolar concentration, for example, as low as about 200 μM, about 150μM, about 100 μM, about 10 μM, about 1 μM, about 100 nM, about 10 nM, orabout 1 nM.

In one embodiment, the methods for small molecule detection providedherein are rapid and can be completed in about 5 minutes to about 120minutes, about 6 minutes to about 110 minutes, about 7 minutes to about100 minutes, about 8 minutes to about 90 minutes, about 9 minutes toabout 80 minutes, about 10 minutes to about 70 minutes about 15 minutesto about 60 minutes, about 20 minutes to about 50 minutes, or about 25minutes to about 40 minutes. In some embodiments, the method can becompleted in about 5 minutes, about 10 minutes, about 15 minutes, about20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about40 minutes, about 45 minutes, or about 50 minutes.

In another embodiment, the methods for small molecule detection providedherein are rapid and can be completed in about 5 seconds to about 5minutes, about 10 seconds to about 4 minutes, about 15 seconds to about3 minutes, about 20 seconds to about 2 minutes, or about 25 seconds toabout 1 minute.

In one embodiment, the subject invention provides a method for detectingsmall molecules that are biomarkers for diagnosis of a disease orcondition, or monitoring therapeutic response to specific treatments. Inspecific embodiments, the condition can be, for example, cancer, aninjury, an inflammatory disease or a neurodegenerative disease. In someembodiments, the condition can be substance abuse, psychosis,schizophrenia, Parkinson's disease, attention deficit hyperactivitydisorder (ADHD), and pain. In some embodiments, the pain is acute painor chronic pain. In some embodiments, the pain is neuropathic pain,e.g., chronic neuropathic pain.

In one embodiment, the methods, assays and products according to thesubject invention can be used for the sensitive and accurate detectionof small-molecule targets in fields including environmental monitoring,food safety, law enforcement, medical diagnostics, and public health.

The subject invention encompasses the use of sequences having a degreeof sequence identity with the nucleic acid sequence(s) of the presentinvention. A similar sequence is taken to include a nucleotide sequencewhich may be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the subject sequence. Typically, the similar sequences will comprisethe same or similar secondary structure as the subject nucleic acidaptamer. In one embodiment, a similar sequence is taken to include anucleotide sequence which has one or several additions, deletions and/orsubstitutions compared with the subject sequence.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within 1 or more than 1 standard deviation, per the practice inthe art. Alternatively, “about” can mean a range of up to 20%, up to10%, up to 5%, or up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed. In the context of compositions containingamounts of ingredients where the term “about” is used, thesecompositions contain the stated amount of the ingredient with avariation (error range) of 0-10% around the value (X±10%).

EXAMPLES

Experimental Section

Materials and Methods

Materials

The names and sequences of the DNA oligonucleotides (5′-3′) are listedbelow, where N represents random base; /Bio/ represents biotinmodification; and MB represents methylene blue. All DNA oligonucleotideswere dissolved in PCR quality water. The concentrations of dissolved DNAwere measured using a NanoDrop 2000 spectrophotometer.

Library: (SEQ ID NO: 1)5′-CGA GCA TAG GCA GAA CTT ACG AC(N30) GTC GTA AGA GCG AGT CAT TC-3′cDNA-bio: (SEQ ID NO: 2) 5′-TTT TTG TCG TAA GTT CTG CCA TTT T/Bio/-3′Forward primer (FP): (SEQ ID NO: 3) 5′-CGA GCA TAG GCA GAA CTT AC-3′RP-bio: (SEQ ID NO: 4) 5′-/Bio/GAA TGA CTC GCT CTT ACG AC-3′Reverse primer (RP): (SEQ ID NO: 5) 5′-GAA TGA CTC GCT CTT ACG AC-3′SCA 2.1: (SEQ ID NO: 6)5′-CTT ACG ACC TTA AGT GGG GTT CGG GTG GAG TTT ATG GGG TCG TAA G-3′SCA-SW-40: (SEQ ID NO: 18)5′-CTT ACG ACC TTA AGT GGG GTT CGG GTG GAG TTT ATG GGG T-3′ SCA-SW-34:(SEQ ID NO: 19) 5′-ACC TTA AGT GGG GTT CGG GTG GAG TTT ATG GGG T-3′

Drug standards, including 3,4-methylenedioxypyrovalerone (MDPV),3-fluoromethcathinone (3-FMC), 4-methylmethcathinone (4-MMC),4-fluoromethcathinone (4-FMC), alpha-pyrrolidinopentiophenone (α-PVP),butylone, cathinone, ethylone, methcathinone, methylone, naphyrone,pentylone, mephedrone, methedrone,3,4-methylenedioxy-α-pyrrolidinobutiophenone,4-methyl-a-pyrrolidinobutiophenone, and4′-methyl-α-pyrrolidinohexanophenone were purchased from CaymanChemicals. Acetaminophen, amphetamine, benzocaine, caffeine, cocaine,diethylthiatricarbocyanine (Cy7), ephedrine, lidocaine, methamphetamine,procaine, promazine, pseudoephedrine,2,2′-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS),chlorpromazine HCl, cocaine HCl, diphenhydramine HCl, levamisole HCl,lidocaine HCl, methamphetamine HCl, promazine HCl, scopolamine HCl, andsucrose and all other chemicals were purchased from Sigma-Aldrich unlessotherwise noted. TOPO TA cloning kit and the plasmid extraction kit(PureLink® Quick Plasmid Miniprep Kit) were purchased from Invitrogen.Tween 20, 30% hydrogen peroxide (H₂O₂) and formamide were purchased fromFisher Scientific. 500 μL micro-gravity columns were purchased fromBio-Rad. Streptavidin-coated agarose resin (capacity: 1-3 mgbiotinylated BSA/ml resin), One Shot Chemically Competent Escherichiacoli, and SYBR Gold were purchased from Thermo Scientific. GoTaq HotStart Colorless Master Mix was purchased from Promega. 3 KDa 3 kDacut-off spin filters were purchased from Millipore. All syntheticcathinones were purchased as hydrochloride salts.

SELEX Strategy

The isolation of aptamers was carried out following alibrary-immobilized SELEX protocol with the parallel-and-serialselection strategy. Detailed information regarding the conditions foreach round of selection are listed in Table 1. The whole aptamerisolation process consists of 5 (ethylone and butylone) or 9 (α-PVP)rounds of parallel selection and 2 cycles of serial selection.

TABLE 1 Detailed information regarding the conditions for each round ofselection Pool size Round # (pmole) Counter-targets Targets Parallel P11000  N/A Butylone (1000 μM) selection 1000  Ethylone (1000 μM) 1000 α-PVP (1000 μM) P2 358 COC (100 μM) Butylone (500 μM) 346 Ethylone (500μM) 368 α-PVP (500 μM) P3 321 COC & PRO (100 μM each) Butylone (500 μM)362 Ethylone (500 μM) 345 α-PVP (500 μM) P4 319 COO, PRC, & LDC (100 μMeach) Butylone (250 μM) 367 Ethylone (250 μM) 292 α-PVP (250 μM) P5 300COC, PRC, & LDC (100 μM each) Butylone (250 μM) 300 Ethylone (250 μM)300 α-PVP (250 μM) P6 300 COC, PRC, & LDC (100 μM each) α-PVP (100 μM)P7 300 COC, PRO, & LDC (100 μM each) α-PVP (100 μM) P8 300 COC, PRC, &LDC (100 μM each) α-PVP (100 μM) P9 300 COC (300 μM) PRC (300 μM) LDC(300 μM) α-PVP (100 μM) Serial S1  300* COC (500 μM) PRC (500 μM) LDC(500 μM) Butylone (100 μM) selection S2 300 Ethylone (100 μM) S3 300α-PVP (100 μM) S4 300 EPH, PSE, ACM, COC, PRC, & LDC Promazinc (500 μM)Butylone (100 μM) S5 300 METH, & AMP (1000 μM each) Ethylone (100 μM) S6300 (500 μM each) α-PVP (100 μM) *Consists of 100 pmole each of poolenriched with butylone (Round P5), ethylone (Round P5) and α-PVP (RoundP9). Acetaminophen (ACM), amphetamine (AMP), cocaine (COC), ephedrine(EPH), lidocaine (LDC), methamphetamine (METH), procaine (PRC),pseudoephedrine (PSE).Parallel Selection

For parallel selections (Round P1-P5 for ethylone and butylone, RoundP1-P9 for a-PVP), three initial pools, each consisting of 1000 pmole DNAlibrary, were used for the three different selection targets (α-PVP,ethylone, butylone). Positive selection was performed with progressivelydecreasing target concentrations (Round P1: 1000 μM, Round P2-P3: 500μM, Round P4-P5: 250 μM for α-PVP, ethylone and butylone; Round P6-P9:100 μM only for α-PVP) to increase selection stringency for enrichingstrong binders. Counter-selection was also performed for each roundprior to positive selection, except the first round, to eliminatenon-specific binders. The number of counter targets and theconcentrations of counter targets were progressively increased duringthe counter selection process to increase selection stringency.Specifically, from Round P1-P5 for ethylone and butylone pools and RoundP1-P9, 100 μM each of cocaine, procaine, and/or lidocaine as a mixturewas used, except for Round P9 for the α-PVP pool which employed 300 μMof each aforementioned counter target employed consecutively.

Serial Selection

Once measurable binding affinity was observed for each pool to theirrespective target after parallel selection, 100 pmole of each individualpool was combined to generate the initial pool for serial selection. Twocycles of serial selection (Cycle 1: Round S1-S3, Cycle 2: Round S4-S6)were performed to specifically isolate cross-reactive aptamers. For eachcycle, positive selection was performed by alternating the selectiontarget (Round S1 & S4: butylone, Round S2 & S5: ethylone, and Round S3 &S6: α-PVP). Target concentration was maintained at 100 μM for each roundof serial selection for maximum selection stringency to isolate highaffinity aptamers. In the first cycle of serial selection, counter-SELEXwas performed by consecutively challenging the pool with a mixture of500 μM each of cocaine, procaine and lidocaine while in the second cycleof serial selection, counter-SELEX was performed by sequentialchallenging with a mixture of 500 μM each of ephedrine, pseudoephedrine,acetaminophen, methamphetamine, amphetamine; a mixture of 1000 μM eachcocaine, procaine, and lidocaine; and 500 μM promazine. From secondround to fourth round in parallel selections, approximately 300 pmole ofenriched library pool for each target from the previous round wereemployed in the subsequence round.

SELEX Procedure

The initial ssDNA library used for SELEX consisted of approximately6×10¹⁴ oligonucleotides. Each library strand is stem-loop structured and73 nucleotides in length, with an 8-base-pair stem flanked by two fixedprimer regions and a randomized 30 nucleotide loop (library: SEQ ID NO:1). For the first round of SELEX, 1000 pmole of library was mixed withbiotinylated capture strands (cDNA-bio: SEQ ID NO: 2) at a molar ratioof 1:5 in selection buffer (10 mM Tris-HCl, 0.5 mM MgCl₂, 20 mM NaCl, pH7.4), heated at 95° C. for 10 minutes, and cooled at room temperaturefor over 30 minutes to ensure hybridization between library and capturestrands. A micro-gravity column was prepared by adding 250 μL ofstreptavidin-coated agarose beads followed by washing of the column with250 μL of selection buffer for three times. A 250 μL solution containingthe library pool was then flowed through the micro-gravity column threetimes in order to conjugate the library to the agarose beads. The columnwas subsequently washed 10 times with selection buffer. Then, 250 μL oftarget (α-PVP, ethylone or butylone) dissolved in selection buffer wasadded to the column, and the eluent was collected. This process wasrepeated an additional two times, and all eluents were combined together(750 μL total).

The resulting pool was concentrated via centrifugation using a 3 KDacut-off spin filter. The concentrated pool (100 μL) was then mixed with1 mL of GoTaq Hot Start Colorless Master Mix with 1 μM forward primer(SEQ ID NO: 3) and 1 μM biotinylated reverse primer (RP-bio: SEQ ID NO:4) to amplify the pool via PCR. Amplification was performed with thefollowing program: 1 cycle of 95° C. for 2 minutes; 13 cycles of 95° C.for 15 seconds, 58° C. for 30 seconds, and 72° C. for 45 seconds; and 1cycle of 72° C. for 5 minutes using a BioRad C1000 thermal cycler.Amplification of the enriched pool and the absence of byproducts wasconfirmed using 3% agarose gel electrophoresis.

To generate single-stranded DNA from the resulting double-stranded PCRproducts, a fresh micro-gravity column was prepared containing 250 μLstreptavidin-coated agarose beads. The amplified pool was then flowedthrough the column three times to conjugate the pool to the beads.Afterwards, the column was washed six times with 250 μL of separationbuffer (10 mM Tris-HCl, 20 mM NaCl, pH 7.4). The column was then capped,and 300 μL of a 0.2 M NaOH solution was added to the column andincubated for 10 minutes to generate single-stranded DNA and then theeluent was collected. An additional 100 μL of 0.2 M NaOH was added toelute residual library strands from the column and this eluent wascollected. Both eluents were combined and neutralized with 0.2 M HCl andthe pool was concentrated via centrifugation with a 3 KDa cut-off spinfilter. The same protocol was performed for subsequent rounds ofpositive selection.

From the second round, counter-SELEX was performed before the targetelution step. Specifically, the library-immobilized column was washedwith 250 μL of counter-target(s) in selection buffer to removenon-specific DNA strands. This process was repeated three (Round: P1-P3and S1-S6) or ten (Round: P4-P5 for ethylone and buthylone, P4-P9 fora-PVP) times. Afterwards, the column was washed 30 times with selectionbuffer to wash away non-specific binders in preparation for positiveselection.

Gel Elution Assay

The enrichment and target affinity, specificity, and cross-reactivity ofthe pools after Rounds P5, P9, S3, and S6 were evaluated using amodified version of a previously reported gel elution assay.Specifically, 50 pmole of enriched library was incubated with 250 pmoleof biotinylated cDNA in 125 μL of selection buffer, heated at 95° C. for10 minutes, and cooled at room temperature for over 30 minutes to annealboth strands. The cDNA-library complex was then mixed with 125 μL ofstreptavidin-coated agarose beads in a microcentrifugation tube andwashed with 625 μL of selection buffer for five times using anend-over-end rotator for 5 minutes followed by centrifugation andremoval of the supernatant. The volume of the library-immobilized beadsolution was adjusted to 150 μL with selection buffer and aliquoted into7 tubes (20 μL per tube). Afterwards, 50 μL of varying concentrations ofthe target (final concentrations: 0, 10, 50, 100, 250, 500, 1000 μM) wasadded into each tube. After rotating for 60 minutes on an end-over-endrotator, the beads were settled by centrifugation and 40 μL of thesupernatant, which contained the target-eluted strands with aconcentration of “c_(s)”, was collected and set aside. Meanwhile theleftover solution (30 μL) was mixed with 50 μL of a 95% formamidesolution containing 10 mM EDTA and incubated at 90° C. for 10 minutes tocompletely release all DNA strands from the beads.

The resulting solution contained both leftover target-eluted strands andnon-target-eluted strands, the concentration of this solution beingtermed as “c_(b)”. The concentration of the target-eluted aptamersolution (c_(s)) and formamide-treated library solution (c_(b)) wereanalyzed via 15% denaturing polyacrylamide gel electrophoresis (PAGE)and the concentrations of the strands were determined via standardizedconcentrations of ladder loaded in the gel. The elution percentage wascalculated using the equation

$\theta = {\frac{V_{1} \times c_{s}}{{V_{2} \times c_{s}} + {V_{3} \times c_{b}}} \times 100\%}$where ⊕ is the fraction of target-eluted strands, V₁ is the volume ofsolution before collection of the supernatant (V₁=62 μL, the volumeoccupied by agarose beads was estimated as 8 μL), V₂ is the volume ofthe collected supernatant containing target-eluted strands (40 μL), andV₃ is the volume of solution after addition of formamide (80 μL).

The same protocol was used to determine the target-cross-reactivity andspecificity of the enriched pool affinity for other synthetic cathinones(3-FMC, 4-MMC, 4-FMC, cathinone, MDPV, methcathinone, methylone,naphyrone, and pentylone) or counter-targets (acetaminophen,amphetamine, cocaine, ephedrine, lidocaine, methamphetamine, procaine,promazine, and pseudoephedrine).

Cloning and Sequencing

Cloning and sequencing of the final enriched pool was performed using apreviously reported protocol. Briefly, the enriched sequences from thefinal pool (Round S6) were amplified by PCR with unlabeled forward andreverse primers (FP: SEQ ID NO: 3 and RP: SEQ ID NO: 5) using the sameprogram as described above in ‘SELEX Procedure’.

At the end of the amplification protocol, an additional 30-minuteextension step at 72° C. was performed to add an A-tail. The PCR productwas cloned into a plasmid vector and transformed into E. coli cellsusing the TOPO TA cloning kit (Invitrogen) according to the supplierrecommendations. The plasmids from 50 randomly picked colonies wereextracted using a PureLink Quick Plasmid Miniprep Kit (Invitrogen) andsequenced at the Florida International University DNA Core Facility. Thesequences of the plasmid and primers were removed, and the resultingaptamer sequences were aligned with the BioEdit software and theconsensus sequence was identified using WebLogo.

Isothermal Titration Calorimetry (ITC)

ITC was perforated using a MicroCal ITC200 instrument (Malvern). All ITCexperiments were carried out using the following protocol at 23° C. Asolution containing the aptamer (final concentration 20 μM) was preparedin selection buffer (10 mM Tris-HCl, 0.5 mM MgCl₂, 20 mM NaCl, pH 7.4)and loaded into the ITC sample cell. Then, 0.35 mM of MDPV or othertargets in the same buffer was loaded into the syringe and titrated intothe cell with an initial 0.4 μL purge injection followed by 19successive 2 μL injections. A spacing of 180 seconds was used betweeneach titration. The final molar ratio between the target and aptamer was3.8:1. The heat generated from each titration was recorded and thebinding enthalpy (ΔH), entropy (ΔS), and dissociation constant (K_(D))were obtained by fitting the resulting titration curve with asingle-site binding model using the MicroCal analysis kit integratedinto Origin 7 software.

Characterization of Cy7 Binding to the Synthetic-Cathinone-BindingAptamer (SCA2.1)

8 μL of different concentrations of SCA2.1, 8 μL of 20 μM Cy7 (finalconcentration 2 μM), and 64 μL of reaction buffer (final concentration10 mM Tris-HCl, 0.5 mM MgCl₂, 20 mM NaCl, 0.01% Tween 20, and 1% DMSO,pH 7.4) were mixed in the wells of a 384-well plate. UV-vis spectra wereimmediately recorded from 450 nm to 900 nm using a Tecan Infinite M1000PRO microplate reader at room temperature. The absorbance value at 785nm (λmax of Cy7 monomer) was plotted against the concentration of addedaptamer. The K_(D) was estimated by non-linear fitting using theLangmuir equation.

Cy7-Displacement Assay for Colorimetric Detection of SyntheticCathinones

8 μL of SCA2.1 (final concentration: 3 μM), 8 μL of Cy7 (finalconcentration 2 μM), 8 μL of varying concentrations of α-PVP, butylone,or ethylone, and 56 μL of reaction buffer (10 mM Tris-HCl, 0.5 mM MgCl₂,20 mM NaCl, 0.01% Tween 20, 1% DMSO, pH 7.4) were mixed in the wells ofa 384-well plate. UV-vis spectra were immediately recorded from 450 nmto 900 nm using a Tecan Infinite M1000 PRO microplate reader at roomtemperature. The absorbance ratio between 670 nm and 785 nm (A₆₇₀/A₇₈₅)were calculated for each sample and signal gain was calculated by(R−R₀)/R₀, where R₀ and R represent A₆₇₀/A₇₈₅ without and with targets,respectively. Using the same protocol, the cross-reactivity andspecificity of the assay were tested using other synthetic cathinones(naphyrone, MDPV, pentylone, methylone, 4-MMC, 4-FMC, 3-FMC,methcathinone and cathinone) and interferents (amphetamine,methamphetamine cocaine, pseudoephedrine, ephedrine, procaine,lidocaine, benzocaine, caffeine, acetaminophen, and sucrose) at aconcentration of 50 μM. Cross-reactivity was calculated using the signalgain of 50 μM α-PVP as 100%. For visual synthetic cathinone detection,the assay was performed using the same protocol but with optimizedconcentrations of SCA2.1 (5 μM) and Cy7 (3.5 μM) with 50 μM of theaformetioned synthetic cathinones or interferents. Photographs of thesamples were taken using a digital camera immediately after mixing allreaction components.

Fabrication of an Electrochemical Aptamer-Based (E-AB) Sensor forDetection of Synthetic Cathinones

The E-AB sensor was fabricated by using polycrystalline gold diskelectrodes (3 mm diameter; BAS). The electrodes were polished andcleaned by following a previously published protocol (ref). The cleangold electrode was incubated with different concentrations of 5′thiolated, 3′ methylene blue-modified SCA-SW-40 or SCA-SW-34 containing2 mM tris-(2-carboxyethyl) phosphine hydrochloride in phosphate-bufferedsaline (PBS) (10 mM phosphate, 1 M NaCl, and 1 mM MgCl₂, pH 7.2) for 12hours. The surface was then rinsed with deionized water and passivatedwith 3 mM 6-mercaptohexanol in the same PBS buffer for 2 hours. Theelectrode was incubated in a Tris buffer (10 mM Tris, 1 mM NaCl, and0.03 mM MgCl₂, pH 7.4) for 1 hour prior to the measurements. Sensorperformance was evaluated by monitoring the electrode in optimizedbuffer containing different synthetic cathinones and interferents usingsquare wave voltammetry (CH Instruments).

Example 1—Parallel-and-Serial SELEX Strategy to Identify Cross-ReactiveAptamers

A rationally designed ‘parallel-and-serial’ SELEX strategy was developedto isolate cross-reactive aptamers for structurally-similar compounds.The strategy has three elements that are crucial for its success. First,the targets used for SELEX are highly important, as target selectiondefines the core structure that is to be recognized by the isolatedaptamer and also creates selection pressure for isolating aptamers thatare insensitive to the identity of peripheral substituents. Thus, asmany targets as needed are chosen that have variations at all desiredsubstituent sites while having the same core molecular framework of thetarget family. Second, the strategy utilizes ‘parallel selection’, inwhich multiple aptamer pools are enriched using each individual target.Cross-reactive aptamers recognizing the shared core structure would beenriched in each of these pools, while aptamers specific to anindividual target should only be enriched in their respective pool.Therefore, when all pools are combined after a few rounds of parallelselection, cross-reactive aptamers should be highly enriched, thusincreasing the likelihood of successful isolation. Third, the combinedpool must then be subjected to ‘serial selection’ with each targetsequentially, a process that ultimately retains only those aptamers thatbind to the core structure shared by these targets.

This strategy was used to isolate a cross-reactive aptamer binding tosynthetic cathinones, a family of designer drugs that share the samechemical core structure with four substituent sites (FIG. 1A). Syntheticcathinones are highly addictive central nervous system stimulants, andare associated with many severe psychological and physiological healthconsequences. Assay development for the detection of these drugs lagswell behind the emergence of new molecules into the market, with currentimmunoassays only capable of detecting a few members.

Specifically, parallel selection was first performed to enrichcross-reactive synthetic-cathinone-binding aptamers using three targets:α-PVP, ethylone, and butylone (FIG. 1A). These targets share the samebeta-keto phenethylamine core structure but have variation at allsubstitution sites typical of the synthetic cathinone family. Parallelselection was performed using three different initial library pools,with one pool being challenged with α-PVP, one with ethylone, and onewith butylone (FIG. 1B).

During the first round, each initial library pool was challenged with1000 μM of target, and eluted strands were collected and amplified forthe next round of selection. To ensure that the aptamer has highspecificity, counter-SELEX was performed from the beginning of thesecond round against structurally-similar non-cathinone moleculesincluding acetaminophen, amphetamine, cocaine, ephedrine, lidocaine,methamphetamine, procaine, promazine, and pseudoephedrine.

In round two, counter-SELEX was first performed for each pool against100 μM cocaine, with positive selection then being performed with 500 μMtarget, as reducing target concentration increases selection stringency.In the third round, the same target concentration was used but anadditional counter-target (100 μM procaine) was included. In rounds fourand five, counter-SELEX was performed against cocaine, procaine, andlidocaine each at a concentration of 100 μM in a mixture with 250 μMtarget for positive selection. After the fifth round, a gel elutionassay was utilized to determine the target-binding affinity of each poolto their respective target. The fraction of eluted library increasedwith increasing target concentrations for the ethylone (FIG. 2A) andbutylone (FIG. 3A) pools, showing that aptamers binding to these targetshad been enriched through parallel selection. Meanwhile, target elutionremained low and constant for the α-PVP pool (FIG. 4A) regardless of theemployed concentration of target, which indicated that the pool was notyet enriched.

The cross-reactivity and specificity of the three pools were furtherdetermined via the gel-elution assay. Both the enriched ethylone andbutylone pools were able to bind to ethylone and butylone as well asprocaine, with the butylone pool capable of also binding to cocaine.Moreover, neither pool displayed any affinity for lidocaine. However,both pools did not demonstrate any binding towards α-PVP (FIGS. 2B and3B), which indicated that the population of highly cross-reactiveaptamers were relatively low. Meanwhile, the α-PVP pool showed noaffinity for any of the targets or counter-targets (FIG. 4B).

Given that the α-PVP pool was not yet enriched, further rounds ofselection were performed. From rounds six to eight, the samecounter-target concentrations from round five was used with afurther-reduced α-PVP concentration of 100 μM for positive selection.For the ninth round, the same α-PVP concentration was used but with 300μM of each of the three aforementioned counter-targets. After the ninthround, the gel elution assay was performed with the enriched pool and aclear target-concentration-dependent elution profile was observed forα-PVP with an estimated dissociation constant (K_(D)) of 28 μM viafitting with the Langmuir equation (FIG. 5A). Notably, only 30% of thelibrary was eluted, even in the presence of 1000 μM α-PVP, indicatingthat a small population of binders was enriched. The cross-reactivityand specificity of this enriched pool were also determined and the pooldisplayed affinity to ethylone and butylone, but was less responsetowards any other interferents (FIG. 5B), which can be attributed to thefact that more rounds of counter-SELEX was performed. Given that thepools enriched with individual targets also cross-reacted to othertargets, it is possible that those pools contained highly cross-reactiveaptamers.

Serial selection was then performed to enrich cross-reactive aptamersand exclude aptamers specific to individual targets. Specifically, 100pmole of each enriched pool obtained from parallel selection werecombined and the resulting combined pool was used as the new library.One cycle of serial selection was first performed, wherein the combinedpool was challenged with each target one-by-one for a total of threerounds. For each round of the first cycle, counter-SELEX was initiallyperformed using 500 μM each of cocaine, procaine, and lidocainesequentially and then performed positive selection with 100 μM of eitherbutylone (round 1), ethylone (round 2), or α-PVP (round 3). After thiscycle of serial selection, a gel elution assay was performed todetermine the cross-reactivity and specificity of the resulting pool(FIG. 6).

Results show that the cross-reactivity towards ethylone and butylonesubstantially increased (K_(D)=82 μM and 77 μM, respectively) relativeto the respective individual pools at the end of parallel selection,while affinity towards α-PVP was similar (K_(D)=34 μM) (FIG. 6A). Thespecificity of the pool was high, with minimal affinity towards cocaineand lidocaine and a moderate response (30% cross-reactivity) to procaine(FIG. 6B).

Another cycle of serial selection was further performed with anidentical selection procedure but with twice the concentration of theoriginal counter-targets and additional counter-targets including 500 μMeach of ephedrine, pseudoephedrine, acetaminophen, methamphetamine, andpromazine. After this cycle, the pool binding affinity was evaluated viathe gel elution assay (FIG. 7). More than 70% of the pool can be elutedby 500 μM of each individual target. The pool affinity towards ethyloneand butylone increased by approximately 10-fold (K_(D)=6.9 μM and 9.5μM, respectively), but the affinity towards a-PVP only marginallyincreased (K_(D)=21 μM) (FIG. 7A). At this stage this enriched-poolmostly consisted of cross-reactive aptamers binding to thecore-structure shared by all synthetic cathinones.

To confirm this, the gel elution assay was used to test thetarget-cross-reactivity of the final enriched pool by challenging itwith the original three targets as well as 13 different syntheticcathinones (pyrovalerone, MPHP, 4-MMC, MDPV, methedrone, naphryone,MDPBP, 3-FMC, 4-FMC, pentylone, methcathinone, methylone, and MEPHP)(Chemical structures see FIG. 8A). Of these targets, 14 syntheticcathinones demonstrated target-elution higher than 60% at aconcentration of 50 μM, while the other two β-FMC and methcathinone)showed less elution but were still significantly higher compared tobuffer alone (FIG. 7B). This result showed that the aptamer was capableof recognizing the core structure of synthetic cathinones, while evenbeing tolerant to side-chain substituents that were not encounteredduring SELEX.

To evaluate the target specificity of the enriched pool, the pool waschallenged with 50 μM of the counter targets (Chemical structures seeFIG. 8B) and none of them showed specifically enhanced elution comparedwith buffer alone (FIG. 7B). Therefore, the final enriched pool wascloned and sequenced. The diversity of enriched pool was low, with 30 ofthe 50 clones having an identical sequence (FIG. 9). This consensussequence was identified as a synthetic-cathinone-binding aptamer termedSCA2.1 (SEQ ID NO: 6). SCA2.1 has a stem-loop structure with a9-base-pair stem and a 28-nucleotide loop under the selection bufferconditions at room temperature (FIG. 10). The target-binding affinity ofthis aptamer to the selection targets was then characterized usingisothermal titration calorimetry (ITC). Specifically, a 300 μM solutionof target was titrated into a 20 μM solution of the aptamer, recordedthe heat released by each titration, and integrated the heats togenerate a binding curve. Binding constants were determined using asingle-site model and similar target-binding affinities (K_(D)) of 2.0μM, 1.1 μM, and 3.1 μM and binding stoichiometries (N) of 1.6, 1.2, 1.7were obtained for butylone, ethylone, and α-PVP, respectively (FIG. 11).These results not only demonstrate the high cross-reactivity of theaptamer, but also show that the binding mechanism may not be explainedby a single-site model. Given the appearance of a two-phase bindingmodality, the aptamer may have a preference for one enantiomeric formsof targets.

Example 2—Naked-Eye Detection of Synthetic Cathinones

SCA2.1 was then used for sensitive, colorimetric detection of syntheticcathinones via a diethylthiotricarbocyanine (Cy7)-displacement assay.Cy7 is a small-molecule dye that exists in equilibrium between monomerand dinner forms, which have absorbance peaks at 760 nm and 670 nm,respectively. Previous studies have shown that Cy7 monomer can bind intohydrophobic target-binding domains of aptamers, which results in asignificant enhancement of absorbance at 760 nm. The binding of targetto the aptamer can displace Cy7 monomer from the binding domain withinseconds, which causes the dye to dimerize in aqueous solution. Thisresults in the reduction of absorbance at 760 nm and enhancement ofabsorbance at 670 nm, which enables Cy7 to be used as a colorimetricindicator for small molecule detection. (FIG. 12A). Such an assay can beemployed to detect synthetic cathinones using SCA2.1.

Specifically, the binding affinity of Cy7 to SCA2.1 was determined bytitrating different concentrations of the aptamer to 2 μM Cy7 (FIG. 13).Increasing the amount of aptamer progressively enhanced the absorbanceof Cy7 monomer at approximately 760 nm, which indicated binding of Cy7to the aptamer (FIG. 13A). A gradual peak shift from 760 nm to 775 nmwas also observed, which is consistent with previous studies that showedthat absorbance of the monomer can change in differentmicroenvironments, such as when the dye binds to the aptamer. Using Cy7absorbance at 775 nm, a K_(D) of 1.6 μM was obtained (FIG. 13B).

Whether the synthetic cathinone targets can efficiently displace Cy7from SCA2.1 was then investigated. Different concentrations of butylonewere first titrated into a mixture of 2 μM Cy7 and 3 μM SCA2.1, andincreasing concentrations of butylone progressively reduced theabsorbance of Cy7 at 775 nm while enhancing the absorbance at 670 nm(FIG. 14A). This change in the absorbance spectra of the dye can beattributed to dimerization of Cy7 monomer when displaced from theaptamer into solution. Based on the absorbance ratio between 670 nm and775 nm (A₆₇₀/A₇₇₅), signal gain was calculated to generate a calibrationcurve displaying a linear range of 0-10 μM and a measurable detectionlimit of 200 nM (FIG. 12B). The same experiment was further performedusing ethylone and α-PVP and similar spectral changes (FIGS. 14B and C),calibration curves, linear ranges, and detection limits (FIG. 12B) wereobtained, indicating the high cross-reactivity of SCA2.1. TheCy7-displacement assay is also compatible with biosamples such as urineand saliva, since the absorbance range of Cy7 is far from the backgroundabsorbance regions of these matrices. Calibration curves were obtainedusing ethylone as target spiked in 50% urine (FIG. 15) and 50% saliva(FIG. 16) with a linear range of 0-10 μM and a measurable detectionlimit of 60 nM and 120 nM, respectively.

The enhanced sensitivity of the assay in these biomatrices can bepossibly attributed to the higher ionic strength of the media, which mayenhance target-binding to the aptamer or Cy7 dimerization. This assay isuseful for label-free detection of synthetic cathinones in urine andsaliva.

The cross-reactivity of the Cy7-displacement assay was further testedfor nine other synthetic cathinones including naphyrone, MDPV,pentylone, methylone, 4-MMC, 4-FMC, 3-FMC, methcathinone and cathinoneat a concentration of 50 μM. Despite the diversity of the side chainssubstituents, all synthetic cathinones induced a significant signalchange in A₆₇₀/A₇₇₅ (FIG. 17A). Moreover, the aptamer-based assayexhibited high cross-reactivity to all tested synthetic cathinones,ranging from 50% to 150% relative to α-PVP (FIG. 17A). This implies thatSCA2.1 mainly recognizes the beta-keto phenethylamine core structure,and variations in the side chains do not significantly affecttarget-binding affinity. Importantly, the assay has excellentspecificity, as the aptamer does not cross-react to non-syntheticcathinone interferents. The Cy7 displacement assay was used to test 12structurally-similar and -dissimilar interferent compounds includingcommon illicit drugs (amphetamine, methamphetamine and cocaine) andcutting agents found in street samples (pseudoephedrine, ephedrine,procaine, lidocaine, benzocaine, caffeine, acetaminophen and sucrose) ata concentration of 50 μM. Despite many of the interferents containing apartial beta-keto phenethylamine structure, the assay yielded noresponse to them (FIG. 17A), indicating the high specificity of theaptamer.

The broad cross-reactivity of SCA2.1 to the synthetic cathinone familyand its high specificity against interferent compounds makes thisaptamer highly favorable for on-site drug screening. Therefore, theCy7-displacement assay was fine-tuned for label-free synthetic cathinonedetection by using a higher concentration of the dye and aptamer tointensify the target-induced color change for naked-eye observation.

This assay was challenged with the aforementioned 12 synthetic cathinonetargets and 11 interferent compounds at a concentration of 50 μM with3.5 μM Cy7 and 5 μM SCA2.1. In the absence of target, the aptamer-boundCy7 monomer has an absorption peak at 775 nm which is not in the visiblerange, and thus the sample is practically colorless. However, in thepresence of a synthetic cathinone, Cy7 is displaced by the target, whichcauses the Cy7 monomer to dimerize. The Cy7 dimer has an absorption peakat 670 nm, which makes the solution appear as a bright blue color thatcan be easily observed by the naked-eye (FIG. 17B). All syntheticcathinones induced a clear-to-blue color change in the solution withinseconds, while no color change was identified upon addition of theinterferent compounds (FIG. 17B). This result clearly demonstrated thefeasibility of the Cy7-displacement assay for instrument-free on-sitedrug screening applications.

Calibration curve of naked-eye detection of ethylone in theconcentration range of 0.4 μM to 200 μM was obtained (FIG. 18). The bluecolor change can be clearly observed by naked-eye with the ethyloneconcentration above 6.3 μM.

Example 3—Generating Structure-Switching Cross-Reactive Aptamers

SCA2.1 adopts a fully folded structure in the absence of target (FIG.19A). Structure-switching aptamers, which undergo a target-inducedconformational change upon binding, can be readily adopted into variousaptamer-based sensor platforms. To successfully transform the isolatedcross-reactive aptamer into a structure-switching aptamer, anexonuclease-assisted truncation method is used. This strategy involvesthe removal of six nucleotides from the 3′-end of SCA2.1 to generate astructure-switching aptamer SCA-SW-40 (SEQ ID NO: 18) that remains in asingle-stranded state in the absence of target (FIG. 19B), but foldsinto a double-stranded structure in the presence of synthetic cathinonedrugs (FIG. 19C).

Example 4—Electrochemical Detection of Synthetic Cathinones

To perform rapid, sensitive, and interference-free electrochemicaldetection of synthetic cathinones in seized substances, thestructure-switching synthetic-cathinone binding aptamer, SCA-SW-40 (SEQID NO: 18), was adopted into an electrochemical aptamer-based (E-AB)sensing platform.

Seized substances contain various impurities and/or adulterants. This isproblematic for common on-site drug screening methods as theseinterfering molecules produce their own colored products in chemicalspot tests and generate their own Raman spectra that overlap with thetarget spectra when performing Raman spectrometry. This causesdrug-related signals to be masked by interfering molecules, resulting ininconclusive results.

E-AB sensors are an ideal choice for the detection of seized substances,because they are insensitive to sample matrix effects. Common drugimpurities and adulterants do not produce an electrochemical signal atthe typically-employed voltage. Additionally, E-AB sensors have rapidresponse times (seconds-scale), can perform detection with minimalsample preparation requirements, and can be easily miniaturized todetect low-volume samples (microliters), thus allowing for analysis oftrace amounts of substances.

E-AB sensing utilizes surface immobilized aptamers to achieve sensitiveand specific analyte detection. The signaling ability of an E-AB sensoris based on target-induced conformational changes in theelectrode-bound, redox-labeled aptamer. Target binding changes theconformation of the aptamer, modifying the electron transfer efficiencybetween the redox reporter and the electrode surface.

To perform E-AB sensing, SCA-SW-40 was synthesized with a 5′ thiol group(e.g., via a 6-carbon linker) and a 3′ methylene blue redox tag (e.g.,via a 7-carbon linker). The aptamers were immobilized onto the goldelectrode surface via thiol-gold chemistry and backfilled using MCH tofill vacant areas on the electrode surface. In the absence of target,SCA-SW-40 exists in an unfolded state, lifting the redox tag away fromthe electrode surface and minimizing electron transfer (FIG. 20A). Thepresence of a synthetic cathinone drug induces a conformational changethat brings the redox reporter close to the electrode surface (FIG.20B), resulting in greater electron transfer efficiency, and ultimatelya large current increase (FIG. 20C).

SCA-SW-40-modified gold electrodes were prepared in the following way.SCA-SW-40 (in disulfide form) was incubated withtris(2-carboxyethyl)phosphine to reduce the 5′ disulfide bonds. Thethiolated aptamer (50, 100, or 500 nM aptamer) was incubated with a goldelectrode (diameter=3 mm) overnight and then backfilled with 3 mM MCH.The surface coverage of immobilized SCA-SW-40 was found to be 3.2, 7.3,or 12×10¹² molecules/cm² for electrodes modified with 50, 100, or 500 nMSCA-SW-40, respectively (FIG. 21A). Calibration curves were generatedusing methylenedioxypyrovalerone (MDPV) as a model synthetic cathinonetarget (FIG. 21B). Measurements were performed in the original selectionbuffer (10 mM Tris-HCl (pH 7.4), 20 mM NaCl, 0.5 mM MgCl₂). Results showthat electrodes modified with a lower surface coverage of SCA-SW-40 hadhigher sensitivity, with the electrode having a surface coverage of3.2×10¹² molecules/cm² producing the highest signal gain (FIG. 21B).Additionally, the stability of the SCA-SW-40 monolayer was measuredafter 50 consecutive scans. Under all conditions tested, the electrodeswere highly stable with little change in surface coverage (FIG. 21C),demonstrating the capability of E-AB sensing for reusable detection ofsynthetic cathinone drugs.

Example 5—Effect of Salt Concentrations on Synthetic Cathinone Detection

To optimize the concentrations of MgCl₂ and NaCl for improved signalgain, detection of 30 μM MDPV was first performed in the presence ofvarious concentrations of MgCl₂ without any NaCl (FIG. 22A). 5 mM MgCl₂was found to give the highest signal gain and was chosen for furtherNaCl optimization. Increasing the NaCl concentration resulted in areduction of signal gain (FIG. 22B), as such 10 mM Tris-HCl (pH 7.4) and5 mM MgCl₂ was determined to be the optimal buffer conditions for MDPVdetection and were therefore used for all subsequent experiments unlessotherwise specified.

Example 6—Effect of E-AB Surface Coverage on Synthetic CathinoneDetection

Results demonstrated that lower SCA-SW-40 surface coverage results inhigher sensitivity (FIG. 21). The surface coverage was further reducedto achieve improved E-AB sensor performance. Gold electrodes wereincubated overnight with either 10, 25, or 50 nM SCA-SW-40, producingsurface densities of 0.73, 1.5, or 3.0×10¹² molecules/cm², respectively(FIG. 23A). MDPV calibration curves were generated for each electrodeand an optimal surface coverage of 1.5×10¹² molecules/cm² were observed(FIG. 23B).

Example 7—Effect of Backfillers on Synthetic Cathinone Detection

To further improve the sensor performance, the effect of differentbackfillers on sensitivity was studied. After modifying the electrodewith 25 nM SCA-SW-40, the electrode was backfilled with either 3 mM DTT,3 mM MCH, or a combination of 1.5 mM DTT and 1.5 mM MCH. Backfillingwith various chemicals resulted in similar surface coverages for eachelectrode (FIG. 24A). MDPV calibration curves were subsequentlygenerated for each electrode and observed the highest signal gain whenonly MCH was used (FIG. 24B).

Example 8—Effect of Distance Between the Redox Tag and Electrode onSynthetic Cathinone Detection

Optimization of DNA surface coverage, electrolyte concentration, andbackfiller achieve a maximal signal gain of 58% at a concentration of100 μM MDPV. Specifically, SCA-SW-40 possesses a 5′-overhang thatseparates the redox tag and electrode by a large distance, therebypossibly limiting electron transfer efficiency upon target binding.Therefore, the removal of the 5′-overhang may improve sensitivity as themethylene blue tag will be orientated closer to the electrode surface inthe presence of target, facilitating efficient electron transfer. Thus,the 5′-overhang of SCA-SW-40 was truncated to produce SCA-SW-34 (SEQ IDNO: 19) (FIG. 25A) and the sensitivity of the resulting aptamer-modifiedelectrode was determined.

SCA-SW-34 was synthesized with a 5′-thiol (e.g., via a 6-carbon linker)and a 3′ methylene blue redox tag (e.g., via a 7-carbon linker) andimmobilized onto a gold electrode surface. Like SCA-SW-40, SCA-SW-34remains unfolded in the absence of target, orientating the methyleneblue redox tag away from the electrode surface (FIG. 25B). Upon targetbinding, SCA-SW-34 undergoes a target-induced conformational change,bringing the methylene blue tag close to the electrode surface andresulting in a large current increase (FIG. 25C).

The effect of the 5′-overhang of SCA-SW-40 on E-AB sensor performancewas studied. Various concentrations of SCA-SW-34 or SCA-SW-40 wereimmobilized onto the gold electrode surface. The use of 25 or 50 nM ofSCA-SW-34 or SCA-SW-40 produced similar surface coverages (FIG. 26A),indicating that the 5′-overhang has minimal effects on probeimmobilization efficiency and surface coverage.

The sensitivity of each electrode was then tested for detection of 30 μMMDPV at various measurement frequencies (FIG. 26B). Under all appliedconditions, SCA-SW-34 demonstrated greater sensor performance thanSCA-SW-40 (FIG. 26B). The results demonstrated that lower SCA-SW-34surface coverage leads to superior E-AB sensor performance with thegreatest signal gain observed at 1.57×10¹² molecules/cm² (FIG. 26B). Theexperiment was repeated using lower concentrations of DNA for electrodemodification (15 and 25 nM). Similarly, the use of 15 or 25 nM ofSCA-SW-34 or SCA-SW-40 produced similar surface coverages (FIG. 27A),with the lowest surface coverage of SCA-SW-34 (1.02×10¹² molecules/cm²)having the highest signal gain (FIG. 27B). Despite a measurementfrequency of 300 Hz providing the greatest signal gain, a frequency of200 Hz was chosen for all subsequent experiments, as 300 Hz maydestabilize the SCA-SW-34 monolayer.

Example 9—Effect of Buffer Conditions on the Sensitivity of the E-ABSensor

To improve the sensitivity of the E-AB sensor even further, the bufferconditions were optimized for MDPV detection. Na⁺ and Mg²⁺ are cationsthat bind to DNA through electrostatic interactions with thenegatively-charged phosphate backbone. This minimizes negative repulsiveforces and aids in aptamer folding and target binding. Increasing theconcentration of both these ions may improve aptamer affinity for MDPV,allowing for higher sensitivity. However, this may also stabilize theDNA structure in the absence of target, resulting in increasedbackground signal. Thus, an optimal concentration of Na⁺ and Mg²⁺ thatallows for high affinity with minimum background would be preferable.

The concentrations of NaCl and MgCl₂ in the ranges of 0-5 mM NaCl and0-1 mM MgCl₂ were then optimized. A NaCl concentration of 0.1 mM and aMgCl₂ concentration of 0.03 mM was found to provide optimal sensorperformance. A calibration curve was generated for MDPV detection usingthe optimized conditions. Results show that increasing concentrations ofMDPV resulted in an increase in current (FIG. 28A). A detection limit of100 nM MDPV was obtained for MDPV under the final optimized conditions(FIG. 28B).

Example 10—Cross-Reactivity and Specificity of the E-AB Sensor

The cross-reactivity of the E-AB sensor was assessed by challenging thesensor with 25 μM of various synthetic cathinones (α-PVP, ethylone,butylone, naphyrone, MDPV, pentylone, methylone, 4-MMC, 4-FMC,cathinone, 3-FMC, and methcathinone) in the optimized detection buffer.MDPV and pentylone yielded the highest signal gain at approximately 75%and 70% signal gain, respectively. Of the twelve synthetic cathinones, 8of them (a-PVP, ethylone, butylone, naphyrone, MDPV, pentylone,methylone, and 4-MMC) yielded a signal gain above 45%. The other foursynthetic cathinones (4-FMC, cathinone, 3-FMC, methcathinone) producedsignal gains between 20-25% (FIG. 29). These results demonstrate thatthe E-AB sensor has excellent cross-reactivity to multiple members ofthe synthetic cathinone family.

To test the specificity of the E-AB sensor, the sensor was challengedwith interferent molecules including amphetamine (AMP), ketamine (KET),cocaine (COC), methylenedioxymethamphetamine (MDMA), heroin (HER),caffeine (CAF), benzocaine (BEN), lidocaine (LIDO), levamisole (LEV),phenacetin (PHE), acetaminophen (ACM), quinine (QUI), sucrose (SUC),lactose (LAC), and mannitol (MAN) (FIG. 30). All interferent moleculeswere tested at a concentration of 250 μM, and 25 μM MDPV was used as apositive control. All but two interferents produced a minimal signalgain (<5% signal gain) in comparison to a 10-fold lower MDPVconcentration that produced a 75% signal gain. Levamisole and quinineproduced signal gains of 10% and 15%, respectively, which is nonethelessstill adequate for cathinone detection given that the purity of suchdrugs in seized samples range between 10-99%. These results demonstratethe specificity of the E-AB sensor against non-cathinone illicit drugsas well as common cutting agents and adulterants found in seizedsubstances.

We claim:
 1. An aptamer-based sensor comprising a cross-reactive aptamerfor synthetic cathinones, the cross-reactive aptamer comprising SEQ IDNO: 16 or a sequence sharing at least 95% identity with SEQ ID No: 16.2. The aptamer-based sensor according to claim 1, the cross-reactiveaptamer being modified with a reporter label selected from a fluorescentdye, a fluorescent or luminescent nanoparticle, and an affinity tag. 3.An aptamer-based sensor comprising a cross-reactive aptamer forsynthetic cathinones, the cross-reactive aptamer comprising SEQ ID NO:15 or a sequence sharing at least 95% identity with SEQ ID No:
 15. 4.The aptamer-based sensor according to claim 3, the cross-reactiveaptamer being modified with a reporter label selected from a fluorescentdye, a fluorescent or luminescent nanoparticle, and an affinity tag. 5.The aptamer-based sensor according to claim 1, the synthetic cathinonehaving a core structure of

wherein R¹ is selected from the group consisting of hydrogen, alkyl,aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl,alkynyl, alkoxy, haloalkyl, acyl, halogen, and hydroxylalkyl, or R¹,taken together with the carbon atom to which it is attached and anadjacent carbon atom, form a substituted or unsubstituted 5- or6-membered homocyclic or heterocyclic ring; R² is hydrogen or alkyl; andR³ and R⁴ are each independently selected from the group consisting ofhydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, alkenyl, alkynyl, haloalkyl, acyl, halogen, andhydroxylalkyl, or R³ and R⁴, taken together with the nitrogen atom towhich they are attached, form a substituted or unsubstituted 5- or6-membered heterocyclic ring.
 6. The aptamer-based sensor according toclaim 5, the synthetic cathinone being selected from3,4-methylenedioxypyrovalerone (MDPV); α-PVP; pyrovalerone; methylone;pentylone; 3,4-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP);mephedrone; 4-methyl-α-pyrrolidinobutiophenone (MPBP);4′-methyl-α-pyrrolidinohexanophenone (MPHP); naphyrone; methedrone;ethylone; butylone; 4-methylmethcathinone (4-MMC); 4-fluoromethcathinone(4-FMC); 3-FMC; methcathinone; and 4-methyl-α-pyrrolidinobutiophenone(MEPBP).
 7. A method for detecting a synthetic cathinone in a sample,the method comprising contacting the sample with the aptamer-basedsensor of claim 1, the aptamer-based sensor further comprising a dye,and detecting the presence of the synthetic cathinone in the sample by asignal generated upon binding of the synthetic cathinone to the aptamer.8. The method according to claim 7, the dye being Cy7.
 9. The methodaccording to claim 7, the sample being a biological sample or anenvironmental sample.
 10. The method according to claim 9, thebiological sample being selected from blood, plasma, urine, tears,sweat, and saliva.
 11. The method according to claim 7, wherein thesignal generated upon binding of the synthetic cathinone to the aptamercan be observed by the naked-eye.
 12. The method according to claim 7,the synthetic cathinone having a core structure of

wherein R¹ is selected from the group consisting of hydrogen, alkyl,aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl,alkynyl, alkoxy, haloalkyl, acyl, halogen, and hydroxylalkyl, or R¹,taken together with the carbon atom to which it is attached and anadjacent carbon atom, form a substituted or unsubstituted 5- or6-membered homocyclic or heterocyclic ring; R² is hydrogen or alkyl; andR³ and R⁴ are each independently selected from the group consisting ofhydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, alkenyl, alkynyl, haloalkyl, acyl, halogen, andhydroxylalkyl, or R³ and R⁴, taken together with the nitrogen atom towhich they are attached, form a substituted or unsubstituted 5- or6-membered heterocyclic ring.
 13. The method according to claim 12, thesynthetic cathinones being selected from MDPV; α-PVP; pyrovalerone;methylone; pentylone; MDPBP; mephedrone; MPBP; MPHP; naphyrone;methedrone; ethylone; butylone; 4-MMC; 4-FMC; 3-FMC; methcathinone; andMEPBP.
 14. A method for detecting one or more synthetic cathinones in asample, the method comprising contacting the sample with theaptamer-based sensor of claim 1, the aptamer-based sensor furthercomprising a dye, and detecting one or more synthetic cathinones in thesample by determining if a color change occurs, a change in color beingindicative of the presence of one or more synthetic cathinones in thesample.
 15. The method according to claim 14, the one or more syntheticcathinones being selected from MDPV; α-PVP; pyrovalerone; methylone;pentylone; MDPBP; mephedrone; MPBP; MPHP; naphyrone; methedrone;ethylone; butylone; 4-MMC; 4-FMC; 3-FMC; methcathinone; and MEPBP.
 16. Amethod for detecting a synthetic cathinone in a sample, the methodcomprising contacting the sample with the aptamer-based sensor of claim3, the aptamer-based sensor further comprising a dye, and detecting thepresence of the synthetic cathinone in the sample by a signal generatedupon binding of the synthetic cathinone to the aptamer.
 17. The methodaccording to claim 16, the dye being Cy7.
 18. The method according toclaim 16, the sample being a biological sample or an environmentalsample.
 19. The method according to claim 18, the biological samplebeing selected from blood, plasma, urine, tears, sweat, and saliva. 20.The method according to claim 16, wherein the signal generated uponbinding of the synthetic cathinone to the aptamer can be observed by thenaked-eye.